Capsicum variety exhibiting a hyper-accumulation of zeaxanthin and products derived therefrom

ABSTRACT

The present invention is concerned with  Capsicum  plants producing greater than about 0.4% zeaxanthin, by weight in the dried, ripe fruit pod flesh, which plants have been developed from commercially grown  Capsicum  cultivars by plant breeding techniques. Zeaxanthin is the dominant carotenoid in the dried ripe fruit pod flesh, when measured in non-esterified forms. Alternatively, these plants may be characterized as exhibiting a high pigmentation measured as an ASTA value and further characterized by the predominant presence of zeaxanthin. The zeaxanthin derived from these  Capsicum  plants can be used in applications that include nutritional supplements, foods, functional foods, cosmetics, animal feeds, aquaculture feeds, and pharmaceuticals.

FIELD OF THE INVENTION

The present invention is concerned with Capsicum plants producinggreater than about 0.4% zeaxanthin, by weight in dried, ripe fruit podflesh, which plants have been developed from commercially grown Capsicumcultivars by plant breeding techniques. Zeaxanthin is the dominantcarotenoid found in the dried ripe fruit pod flesh, when measured innon-esterified forms. Alternatively, these plants may be characterizedas exhibiting a high pigmentation measured as an ASTA value and furthercharacterized by the predominant presence of zeaxanthin. The zeaxanthinderived from these Capsicum plants can be used in applications thatinclude nutritional supplements, foods, functional foods, cosmetics,animal feeds, aquaculture feeds, and pharmaceuticals.

BACKGROUND OF THE INVENTION

The ripe fruit of Capsicum species are a well-known, important source ofa variety of carotenoids, including oxygenated carotene derivatives,commonly referred to as xanthophylls. Capsicum species containcapsanthin, capsorubin, cryptocapsin, zeaxanthin, lutein, and othercarotenoids that have substantial nutritional and medical value.Epidemiological studies have shown that frequent and regular consumptionof carotenoids reduces risks of chronic disorders, such ascardiovascular diseases [Kohlmeier et al. (1995)] or cancer [Murakoshiet al., (1992); Levy et al. (1995); Tanaka et al., 1994) Ito et al.(2005), Connor et al. (2004), and Rock et al. (2005)]. Carotenoids mayalso function as antioxidants in disease prevention. Both zeaxanthin andlutein are reported to possess strong anti-tumor properties [Packer etal. (1999)]. Epidemiologic studies suggest that the antioxidantpotential of dietary carotenoids may protect against the oxidativedamage that can result in inflammation. A modest increase in dietarycarotenoid intake is associated with a reduced risk of developinginflammatory disorders such as rheumatoid arthritis [Pattison, et al.(2005)].

A higher dietary intake of carotenoids is also associated with a lowerrisk for AMD (Age-related Macular Degeneration) occurring in olderadults. Hereditary forms with an early onset include Stargardts, Best'sDisease and progressive Cone Dystrophy. Hereditary retinal degenerationsthat attack the whole of the retina tend to be more severe. The mostcommon types of these diseases are Retinitis Pigmentosa, Choroideremia,Ushers Syndrome and diabetic retinopathy. Individuals consuming thehighest levels of carotenoids exhibit a 43% (statistically significant)lower risk for AMD [Seddon et al., (1994). The specific carotenoids,zeaxanthin and lutein, are most strongly associated with a reduced riskfor AMD. Zeaxanthin and lutein are the sole xanthophyll pigments foundin the retina and concentrated in the macula. Excellent reviews of therole of carotenoids in the macula are found in Davies, et al., 2004,Stahl et al. (2005), Stringham et al. (2005), Ahmed et al. (2005), Stahl(2005), Beatty et al. (2004), Davies (2004), and Alves-Rodrigues (2004).

There is a strong association between higher consumption of dark greenvegetables, which contain xanthophylls, including zeaxanthin and lutein,and a decreased risk for light-induced oxidative eye damage, such ascataract formation, see Brown et al. (1999) and Ribaya-Mercado (2004).Although dark green vegetables are an excellent dietary source ofzeaxanthin and lutein, the isolation and purification of these compoundsin large quantities from green vegetables is time-consuming and costly.Twenty-five grams of a fresh, dark green vegetable such as kaletheoretically provide 10 mg of lutein. (Khachik et al. 1995). Corn, oneof the highest plant sources of zeaxanthin, contains 0.528 mg ofzeaxanthin per 100 grams of corn (Lutein and Zeaxanthin ScientificReview, Roche Vitamins Technical Publication HHN-1382/0800). It wouldrequire 1.9 kg of corn or 0.623 kg of peppers to provide 10 mg ofzeaxanthin from these sources.

Therefore, a highly concentrated source of natural zeaxanthin is neededfor the manufacture of dietary supplements and functional foods.Moreover, zeaxanthin is an important ingredient to add color to foodsand as an additive in animal feeds to color poultry skin, egg yolks,fish flesh and the like. A natural source of zeaxanthin that can be usedin foods is preferred and/or regulated over a synthetic product in theseapplications.

“GRAS” is an acronym for the phrase Generally Recognized As Safe. Undersections 201(s) and 409 of the Federal Food, Drug, and Cosmetic Act (theAct), any substance that is intentionally added to food is a foodadditive, that is subject to premarket review and approval by FDA,unless the substance is generally recognized, among qualified experts,as having been adequately shown to be safe under the conditions of itsintended use, or unless the use of the substance is otherwise excludedfrom the definition of a food additive. Regardless of whether the use ofa substance is a food additive use or is GRAS, there must be evidencethat the substance is safe under the conditions of its intended use. FDAhas defined “safe” (21 CFR 170.3(i)) as a reasonable certainty in theminds of competent scientists that the substance is not harmful underits intended conditions of use. The specific data and information thatdemonstrate safety depend on the characteristics of the substance, theestimated dietary intake, and the population that will consume thesubstance.

Zeaxanthin derived from natural sources is usually obtained as a mixtureof free xanthophyll compounds together with the pigment in the form ofmixtures of mono and diesters of fatty acids. The fatty acids generallycontain from eight to twenty carbon atoms. Methods for converting theseesterified forms of zeaxanthin to a free alcohol form are well known anddocumented. Methods for preparing esters from the non-esterified formare also known and documented.

Zeaxanthin from natural sources is generally obtained in the form of anall-trans isomer. It is well known that the trans isomer can beconverted to cis forms by the application of heat and/or light or by theaddition of a catalytic amount of iodine (Zechmeister, 1962; Khachik, etal. 1992; Updike et al., 2003; Englert, et al. 1991 and referencestherein; Karrer and Jucker, 1950. Zechmeister also discussesisomerization by acid catalysts, contact with active surfaces, via borontrifluoride complexes and bio-stereoisomerization. Given the number ofdouble bonds in the structure, a large number of different cis isomersare possible. Both cis and trans isomers have been detected in theretina.

Zeaxanthin also exists in two enantiomeric and one meso form, namely3R,3′R; 3S,3′S and 3R,3′S (note 3S,3′R is identical to 3R,3′S). Allthree stereoisomers have been found in the human retina (U.S. Pat. No.6,329,432), but the 3R,3′R isomer is dominant. It is difficult toseparate these three isomers of zeaxanthin from each other in commercialquantities for human consumption. Therefore, for synthetic production ofzeaxanthin, either a chiral process or a chiral separation process isneeded in order to purify and produce the 3R,3′R stereoisomer.

Age-related Macular Degeneration (AMD) is the leading cause of blindnessfor people older than 65 in the United States, and is expected to affect40 million U.S. residents by the year 2030 [Abel, (2004). Treatments toameliorate the effects of the disease and methods for preventing theonset of the disease are desperately needed. Since lutein and zeaxanthinplay a critical role in the protection of the macula, it is importantthat people have access to these compounds, either through dietarysources, through supplements, or through so-called functional foods,which foods contain enhanced levels of these nutrients. Numerousepidemiological studies suggest that the typical intake of lutein andzeaxanthin is only in the 1-3 mg/day range, see Brown et al. (1999) andLyle et al. (1999). Seddon et al. (1994) reported a relationship betweenthe intake of lutein and zeaxanthin at 6 mg per day and a decreased riskof AMD and cataracts. This dietary gap of 3-5 mg per day can beeliminated with the use of supplements.

There is a perceived need in the marketplace for naturally derivedzeaxanthin, as opposed to synthetic zeaxanthin, that can serve as adietary source in the form of a dietary supplement, a food or beverageadditive, or a food or beverage colorant. Furthermore, there is a needfor zeaxanthin for dietary supplements, food or beverage additives, andfood or beverage colorants in biologically available forms.

There is also a need for naturally derived zeaxanthin, as opposed tosynthetic zeaxanthin, that can serve as an additive in animal feeds,such as poultry feed, to color flesh and skin, egg yolks and fish flesh.Certain types of poultry feed additives prepared from corn glutencontain a relatively high percentage of zeaxanthin (about 15-30%), whenmeasured as a percentage of total carotenoids. However, the totalcarotenoid content of these feed additives is very low (only about 100milligrams of total carotenoids per pound of poultry feed). Another typeof poultry feed additive is prepared from marigold extracts. Thisadditive contains roughly 100-200 times as much yellow pigment per poundof additive (i.e., about 10 to 20 grams of lutein and zeaxanthin perpound); however, more than 95% of the yellow pigment in this marigoldpreparation is lutein, not zeaxanthin. Zeaxanthin comprises only about 2to 5% of the yellow pigment in this poultry feed additive (U.S. Pat. No.RE 38,009).

Genetics

The accumulation of carotenoids in Capsicum fleshy fruit is wellstudied, with many known biosynthetic genes cloned, sequenced andfunctionally characterized on some level. Although the majority ofinvestigations into carotenoid biosynthesis has been carried out in themodel system Solanum lycopersicon (tomato), additional work has shown ahigh level of conservation of these genes among all plant speciesaccumulating carotenoids [Hirschberg (2001)]. Also, certain carotenoidsshow taxonomic specificity. For example, capsanthin and capsorubin areresponsible for the red color seen in ripe pods of Capsicum, and are notseen in any other genus. These two carotenoids are synthesized via theaction of capsanthin-capsorubin synthase (Ccs) from antheraxanthin andviolaxanthin respectively. In the absence of Ccs, peppers do notaccumulate significant amounts of capsanthin or capsorubin and theresulting ripe fruit are orange in color [Bouvier, et al., (1994)].

The dietary supplement marketplace in both the US and in Europe does notaccept nutrients that are derived from genetically modified organisms.Therefore, there is a need for a naturally derived zeaxanthin productthat is not derived from a genetically modified plant.

Currently, zeaxanthin is available from a number of sources. It isproduced synthetically, extracted from plant matter and extracted frombacteria.

Synthetic Zeaxanthin

The all-trans 3R,3R′ zeaxanthin isomer is produced synthetically, and aprocess for its production is disclosed in U.S. Pat. No. 4,952,716 andU.S. Pat. No. 5,227,507. Synthetic zeaxanthin is commercially availablefrom DSM, who purchased the technology from Hoffmann-LaRoche.Hoffmann-LaRoche had obtained two patents that describe the chemicalsynthesis of the 3R,3′R isomer of zeaxanthin; these are U.S. Pat. No.4,952,716 and U.S. Pat. No. 5,227,507. Processes disclosed thereinrequire the production and purification of three major intermediates,with yields of approximately 70 to 85% for each intermediate from itsprecursor. The overall process disclosed in these patents apparentlyrequires a series of 14 reaction steps, which take a minimum of 83 hours(excluding purification), and yield a mixture of reactants and products.The final reaction mixture must then be extensively treated to purifythe 3R,3′R isomer of zeaxanthin. Accordingly, the entire processrequired for both synthesis and purification using this technique makesproduction on a commercial scale overly difficult, and expensive. Thezeaxanthin produced synthetically is currently available only in thenon-esterified form. A significant problem with certain syntheticallyderived carotenoids is elevated levels of residual solvents (used intheir synthesis) that typically remain in and contaminate the finalproduct. For example, commercial synthetic beta-carotene was analyzed inour laboratory and shown to contain residual levels of toluene oracetone, of 2000 and 1200 ppm, respectively, depending on the syntheticsource. These levels, generally unknown to the public, are undesirable,and are roughly 50 to 100 times higher than levels of residual solventspermitted under 21 CFR §173 for spice extractives containing high levelsof carotenoids, such as paprika or carrot oleoresin.

Plant Sources of Zeaxanthin

The public generally prefers to consume compounds that are derived fromnatural sources as opposed to those that are produced synthetically.Natural sources containing high levels of zeaxanthin currently includecertain mutant varieties of marigold flower petals, berries of the genusLycium and Physalis, and specifically Chinese wolfberries (Lyciumchinense). U.S. Pat. No. 6,191,293 discloses that preferred materialscontaining zeaxanthin “include fruits like oranges, peaches, papayas,prunes, and mangos.” There is no mention in this patent of the genusCapsicum.

Marigolds

Marigold (Tagetes erecta) petals have a long history as a commercialsource of the carotenoid pigment, lutein. Dried marigold flowers containapproximately 1-1.6% carotenoids by weight and lutein esters generallyaccount for 90% of the total carotenoids (Antony et al., 2001). U.S.Pat. No. 6,784,351 discloses a mutant marigold that expresses zeaxanthinat high levels, where zeaxanthin is the dominant carotenoid pigment.Marigold petals, however, are not a recognized food. Although luteinderived from marigolds has been introduced as a food additive throughthe use of the self-affirmed GRAS (Generally Recognized as Safe)process, it cannot be added to foods if it changes the food's color.This is because lutein is not recognized as an exempt food colorantunder 21 CFR §73.

There is another problem associated with pigments isolated frommarigolds. Marigolds are often planted around gardens because theynaturally produce insecticidal compounds and when planted in proximityto other plants, help shield them from insect predation. One type ofthese natural insecticides is a group of compounds known asterthiophenes and related compounds. Terthiophenes are potent phototoxicagents that cause light-activated damage to biological systems [Downumet al., (1995); Aranson, et al., (1995)]. These phototoxic compounds canbe difficult to separate from marigold-derived zeaxanthin. Analysis ofcommercially available zeaxanthin (and lutein) from marigold sourcesdemonstrates that such preparations contain measurable levels ofphototoxic terthiophenes (see Example 12). Therefore, marigold-derivedzeaxanthin is certainly not a preferred form for eye health.α-Terthiophene (also known as α-terthienyl) and other marigoldconstituents, such as butenylbithiophene and hydroxytremetone have beenreported to have sensitizing properties leading to allergic contactdermatitis [Hausen et al, (1995)]. The zeaxanthin-containing extracts ofthe present Capsicum varieties do not contain these sensitizing orphotosensitizing components.

Conversion of Marigold-Derived Lutein to Zeaxanthin

Lutein to zeaxanthin isomerization reactions have been known for morethan 40 years. One process disclosed in, U.S. Pat. No. 6,376,722 usessodium ethoxide, methanol, potassium methoxide, methyl sulfate, andcombinations thereof to effect this conversion.

The weaknesses of this approach are 1) that zeaxanthin derived frommarigolds is not GRAS for food and 2) that phototoxic compounds derivedfrom marigold are not necessarily removed. Additionally, the extrareaction step is also expensive and lowers the yield of zeaxanthinobtained.

Wolfberries

High concentrations of the dipalmitate ester of zeaxanthin have beenisolated from wolfberries (Lycium chinense) which have a history of usein Chinese medicine, Zhou et al., (1999). Since they are not a GRAS foodsubstance, according to 21 CFR 182, the potential use of wolf berries infood systems is limited.

Fruit and Vegetable Crops

Zeaxanthin is found in a wide variety of fruits and vegetables as shownin Table 1 (Lutein and Zeaxanthin Scientific Review, Roche VitaminsTechnical Publication HHN-1382/0800). These levels are quite lowcompared to the concentrations present in the instant invention [about60,000 micrograms/100 g on a raw (wet) basis].

TABLE 1 Concentration of zeaxanthin in commonly consumed fruits andvegetables. Tangerine, mandarin 142 microgram/100 g 0.000142% Kale(cooked) 173 microgram/100 g 0.000173% Spinach (cooked) 179microgram/100 g 0.000179% Lettuce (cos or romaine, raw) 187microgram/100 g 0.000187% Collard greens (cooked) 266 microgram/100 g0.000266% Turnip greens (cooked) 267 microgram/100 g 0.000267% Spinach(raw) 331 microgram/100 g 0.000331% Corn (frozen, cooked) 375microgram/100 g 0.000375% Persimmons (Japanese, raw) 488 microgram/100 g0.000488% Corn (sweet, yellow, cooked) 528 microgram/100 g 0.000528%Pepper (orange, raw) 1606 microgram/100 g  0.001606%

Capsicum

There are two principle types of Capsicum annuum which have a very lowcapsaicin content: bell and paprika types. The presence or absence ofcapsaicin, the pungent principle in peppers, is not critical to thisinvention, as some paprikas are perceptibly hot.

Three major pigment type classes of paprika-type peppers are discussed,which are herein referred to as reds, oranges, and yellows. Red, orangeand yellow fruit of the Capsicum genus are generally good dietarysources of carotenoids. The pepper referred to in Table 1. is aCapsicum. The appearance of a given class is determined by the relativeamounts of the pigments in combination with the total pigmentconcentration. Regardless of the total concentration, and visualappearance, these classes can be differentiated by spectral analysis,and by HPLC. For example, a pod from a red paprika will appear orange ifit has a low pigment concentration, but has a visible spectrum and HPLCanalysis different from the instant orange paprika exhibiting a highzeaxanthin content. In a red paprika, substantial amounts of the redpigments capsorubin and capsanthin are present. In the orange paprika, aminor amount of these two pigments are present, and a very highconcentration of zeaxanthin is present. In yellows, the two red pigmentsare absent as well as a precursor, violaxanthin. Lutein and other yellowpigments and their precursors are present at significantly higher ratiosto zeaxanthin, and total pigment content is much lower as shown by amuch lower ASTA value.

Table 2. summarizes the concentration of zeaxanthin and the percentageof zeaxanthin relative to total carotenoids in dried Capsicum fruitswhich have been reported in the literature. Table 2 shows that thepercent zeaxanthin with respect to the total carotenoids in theCapsicum, as well as the weight percent of zeaxanthin as a percent ofdry weight of the fruit, are much lower than the surprisingly highamounts of zeaxanthin which is characteristic of the instant invention.

TABLE 2 Content and ratios of zeaxanthin in prior art Capsicum varieties(dry weight). Zeaxanthin as Zeaxanthin % of total as % of drycarotenoids fruit weight Reference 9.15 0.045 Matus et al., (1991) 3.830.06 Almela et al., (1991) 2.91 0.043 ″ 4.35 0.058 ″ 2.07 0.026 ″ 4.320.053 ″ 2.73 0.027 ″ 4.26 0.034 ″ 8.49 0.273 Deli et al., (1992) 0.009Minguez-Mosquera et al., (1993) 0.03 ″ 16.2 0.161 Deli et al., (1996)17.9 0.109 ″ 20.5 0.027 ″ 4.1 0.033 Almela et al., (1996) 3.3 0.044 ″14.2 0.019 Topuz et al., (2003) 13 0.013 ″ 6.3 0.081 Hornero-Mendez etal., (2002) 6 0.045 ″ 8.5 0.064 ″ 8.2 0.056 ″ 7 0.068 ″ 6.9 0.072 ″ 8.40.041 ″ 14 0.135 ″ 14.9 0.165 ″ 7.7 0.074 ″ 9 0.073 ″ 8.4 0.081 ″ 8.10.049 Deli et al., (1997) 17.5 0.0952 Deli et al., (2001) 8.8 0.1145 ″6.2 0.0312 Minguez-Mosquera et al., (1994) 10.9 0.073 ″ 7.2 0.028Muller, H. (1997) 3.1 0.006 Camara et al. (1978) 7.9 0.134Minguez-Mosquera et al (1993) 4.6 0.055 Minguez-Mosquera et al (1993)5.2 0.064 Minguez-Mosquera et al (1994) 9.2 0.045 Biacs et al. (1993)11.3 0.103 Rahman et al (1980) 8.9 0.023 Rahman et al (1980) 8.1 0.02Rahman et al (1980) 9.2 0.02 Rahman et al (1980) 5.28 0.024 Biacs, P. A.et al., (1994). 4.5 Deruere, J., etal. (1994). 2.3 Nys, Y. et al.,(2000) 2.3 Fisher, C. et al., (1987) 6.5 Nys, Y. et al., (2000) 6.5Fisher, C. et al., (1987) 3.1 Nys, Y. et al., (2000) 3.1 Fisher, C. andKocis, J. A. J. Agric. Food Chem. 1987, 35, 55-57. 4 Nys, Y. et al.,(2000) 4 Fisher, C. et al., (1987) 15.67 0.0201 Russo, V. M. et al.,(2002) 11.27 0.0397 Russo, V. M. et al., (2002)Indeed, this fact has been stated by, Breithaupt et al. (2005), whoobserved “additionally, oleoresins containing zeaxanthin as sole or evenmajor xanthophyll are not available.” One of the highest amounts orlevels of zeaxanthin which has been previously described for Capsicumvarieties or cultivars is found in the longum nigrum variety as reportedby Deli et al., (1992). This variety contains about 0.273% zeaxanthin inthe dried, ripe fruit pod flesh. However, the percent ratio ofzeaxanthin to total carotenoids in this longum nigrum variety is only8.49%. Varieties with somewhat higher ratios of zeaxanthin relative tototal carotenoids have been described. The lycospersiciforme rubrumvarieties described by Deli et al., (1996), show percent ratios ofzeaxanthin to total carotenoids of 16.2%, 17.9% and 20.5%; however, themass of zeaxanthin present in dried, ripe fruit pod flesh is much lowerin these varieties, 0.027%, 0.109% and 0.161% of the total dried ripefruit pod flesh, respectively.

There are reports in the literature on the analysis of carotenoids infresh Capsicum fruit. The use of fresh versus dehydrated fruitintroduces a complicating factor into estimating the amount ofzeaxanthin in the fresh fruit for comparison with that amount found indried fruit (as reported in Table 2). Breithaupt et al., (2001) havefound that an orange pepper (Capsicum annuum L. Grossum Grp.) contains9234 micrograms of total carotenoids per 100 grams of fresh fruit podflesh. The results were reported as lutein dimyristate equivalents, andneither the absolute nor relative amounts of zeaxanthin were reported.Most paprika-type peppers have a moisture content of 80-85%. Succulentvarieties (e.g. bell peppers) have been reported to contain up to 92%moisture (Banaras et al., 1994). Applying assumptions in this case toskew the results toward high zeaxanthin content, specifically, that thispepper was a bell pepper with 92% moisture, and, unrealistically, thatzeaxanthin made up all the carotenoids present, the Capsicum sample inquestion would contain only 0.12% zeaxanthin. Weller et al., 2003, found3.03 milligrams of zeaxanthin per 100 grams of a fresh orange pepper(Capsicum annuum L.). Using the previous 92% water content assumption,this calculates to 0.04% zeaxanthin on a dry-weight basis. The ratio ofzeaxanthin to total carotenoids reported by Weller et al. for thisparticular pepper was 44%. These authors also describe a red pepper with16.75 mg of zeaxanthin per 100 g of fresh fruit. Using the same,unrealistic assumption, that this is a bell pepper with 92% watercontent, this calculates to a 0.21% zeaxanthin. The ratio of zeaxanthinto total carotenoids for this red pepper is only 15%.

Abellan-Palazon, et al., 2001, reported a paprika cultivar treated withtitanium ascorbate to have 0.56% zeaxanthin, however, the percentage ofzeaxanthin relative to other carotenoids was only 16.6% for thiscultivar. The water content of this fresh pepper was given as 79.9% andthis was the factor used for the moisture correction. Abellan-Palazon,et al. also reported that after drying this sample, the mass percentzeaxanthin fell to 0.16% and the percentage of zeaxanthin relative toother carotenoids fell to 8.4%. Sommerburg, et al., 1998, report thatorange pepper was the vegetable with the highest amount of zeaxanthinwith 37 mole percent. Adjusting for molecular weights, this calculatesto ˜37.8% by weight, well below the >50% reported in the instantinvention. Sommerburg's data do not allow calculation of the masspercent zeaxanthin in the orange pepper from the mole percent data.

Bacterial Sources of Zeaxanthin

Bacteria provide another source of zeaxanthin as in U.S. Pat. No. RE38,009, which discloses a method to produce zeaxanthin by a fermentationprocess with Flavobacterium multivorum (ATCC 55238). Other bacteria havebeen identified that can express zeaxanthin, and they include microbesfrom the genus Flavobacter (ATCC 21081, 21588, and 11947). Zeaxanthinfrom a bacterial source is not GRAS. Furthermore, the safety of extractsfrom bacteria is not established. No commercial source of bacteriallyderived zeaxanthin is known to be available.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide novel Capsicum genusplants, or regenerable portions thereof, which plants produce fruit podswhich exhibit in their dried, ripe flesh a hyper-accumulation ofcarotenoid pigment, wherein zeaxanthin is a mixture of free zeaxanthinand fatty acid esters, and is the dominant carotenoid when measured innon-esterified forms.

It is an object of the present invention to provide novel Capsicum genusplants, or regenerable portions thereof, which plants produce fruit podswhich exhibit in their dried, ripe flesh a hyper-accumulation ofcarotenoid pigment, wherein zeaxanthin, measured as free diol, ispresent at greater than about 0.4% by weight of the dry, ripe fruit podflesh.

It is an object of the present invention to provide novel andcommercially viable strains of Capsicum annuum paprika type plants,which plant produces orange-colored fruit pods and which plant exhibitsin the dried ripe fruit pod flesh, carotenoid pigments with an ASTAvalue of greater than 175, wherein zeaxanthin is the dominantcarotenoid.

An additional object of the invention is the provision of Capsicumproducts/compositions derived from such plants.

An additional object of the invention is the provision of processes fordeveloping such plants, extracting Capsicum products from the ripe podflesh of such plants and methods of treating various conditions withproducts derived from such plants.

DESCRIPTION OF THE INVENTION

What we therefore believe to be comprised by our invention may besummarized inter alia in the following words: Capsicum variantsdeveloped through a selective breeding process which express highabsolute and relative levels (compared to total carotenoids whenmeasured in non-esterified forms) of zeaxanthin, as a mixture of freezeaxanthin and fatty acid esters of zeaxanthin. The invention relates toCapsicum plants, regenerable portions thereof, hybrids or latergenerations, wherein the dried, ripe fruit pod flesh thereof exhibits alevel of zeaxanthin, as a percentage of dry, ripe fruit pod fleshweight, which is greater than 0.4% measured as total non-esterifiedzeaxanthin following a saponification process. The invention furtherrelates to Capsicum plants, regenerable portions thereof, hybrids orlater generations, wherein the dried, ripe fruit pod flesh thereofexhibits a percentage of zeaxanthin relative to total carotenoids [masszeaxanthin/(mass zeaxanthin plus mass of other carotenoids)×100] whichis greater than 50% when measured in non-esterified forms. The inventionfurther relates to strains of Capsicum annuum paprika type plants, whichplant produces orange-colored fruit pods and which plant exhibits in thedried ripe fruit pod flesh, carotenoid pigments with an ASTA value ofgreater than 175, and by the predominance of zeaxanthin.

BRIEF DESCRIPTION OF THE INVENTION

A plant, or regenerable portion thereof, of the Capsicum genus, whichplant produces fruit pods and which plant exhibits in the dried ripefruit pod flesh, a hyper-accumulation of carotenoid pigment, whereinzeaxanthin is the dominant carotenoid, when measured in non-esterifiedforms, such a

plant, or regenerable portion thereof, which is a member of the speciesannuum, such a

plant, or regenerable portion thereof, which is a paprika variety, sucha

plant, or regenerable portion thereof, wherein the mass of zeaxanthin,when measured in non-esterified form, is greater than 0.4% of the totaldried ripe fruit pod flesh, such a

plant, or regenerable portion thereof, wherein the percentage ofzeaxanthin relative to total carotenoids in the dried ripe fruit podflesh is greater than 50%, such a

plant, or regenerable portion thereof, wherein the mass of zeaxanthin isgreater than 0.4% of the total dried ripe fruit pod flesh, such a

plant, or regenerable portion thereof, of the Capsicum genus which plantproduces fruit pods and which plant exhibits in the dried ripe fruit podflesh, zeaxanthin, and wherein the mass of zeaxanthin, when measured innon-esterified form, is greater than 0.6% of the total dried ripe fruitpod flesh, such a

plant, or regenerable portion thereof, wherein the mass of zeaxanthin isgreater than 0.7% of the total dried ripe fruit pod flesh, such a.

plant, or regenerable portion thereof, wherein the mass of zeaxanthin isgreater than 0.8% of the total dried ripe fruit pod flesh, such a

plant, or regenerable portion thereof, wherein the mass of zeaxanthin isgreater than 0.9% of the total dried ripe fruit pod flesh, such a

plant, or regenerable portion thereof, which plant exhibits in the driedripe fruit pod flesh, an ASTA value greater than 175, wherein zeaxanthinis present at a level of greater than about 50% of the HPLC area countof the total pigments, such a

plant, or regenerable portion thereof, which plant exhibits in the driedripe fruit pod flesh, an ASTA value greater than 200, such a

plant, or regenerable portion thereof, which plant exhibits in the driedripe fruit pod flesh, an ASTA value greater than 225, such a

plant, or regenerable portion thereof, which plant exhibits in the driedripe fruit pod flesh, an ASTA value greater than 275, such a

plant, or regenerable portion thereof, wherein the mass of zeaxanthin isgreater than 0.4% of the total dried ripe fruit pod flesh, such a

plant, or regenerable portion thereof, characterized by a capsanthinplus capsorubin content of less than about 10% of the HPLC area count oftotal pigments, such a

plant, or regenerable portion thereof, characterized by a capsanthinplus capsorubin content of less than about 7% of the HPLC area count oftotal pigments, such a

plant, or regenerable portion thereof, characterized by a zeaxanthincontent of greater than about 60% of the HPLC area count of totalpigments, such a

plant, or regenerable portion thereof, characterized by a zeaxanthincontent of greater than about 70% of the HPLC area count of totalpigments, such a

an oleoresin composition derived from the plant, or regenerable portionthereof, such a

plant, or regenerable portion thereof, wherein the regenerable portionis selected from the group consisting of embryos, meristems, pollen,leaves, anthers, ovules, roots, root tips, fruit pods, seeds, petals,flowers, fibers, bolls, and protoplasts or callus derived therefrom,such a

cell culture or tissue culture of the plant, or regenerable portionthereof, such a

grafted plant or progeny of the regenerable portion, such a

seed, which on planting in a suitable environment and grown to maturityyields a plant of the Capsicum genus, such a

hybrid Capsicum plant, wherein one ancestor is a Capsicum variety, sucha

genome of the plant, or regenerable portion thereof, such a

plant extract composition comprising zeaxanthin derived from theCapsicum plant, or regenerable portion thereof, such a

plant extract composition which is an ingredient in cosmetics andcleaning preparations selected from lipsticks, lotions, soaps,foundations, mascara, eye shadow, body scrubs, sun lotion, muds, packs,masks, shampoos, conditioners and toothpastes, such a

plant extract composition which is an ingredient in animal feedsupplements, such a

plant extract composition which is an ingredient in foods and beverages,such a

plant extract composition which is a colorant in foods, beverages, andanimal feed, such a

plant extract composition wherein the zeaxanthin is in the form ofmono-esters, di-esters, the free alcohol form, or a combination thereof,such a

plant extract composition wherein the zeaxanthin is an all transgeometric isomer, or cis geometric isomers or combinations thereof, sucha

plant extract composition in the form of a solid or a semi-solid, such a

plant extract composition wherein the form is selected from powders,beadlets, water-dispersible powders, crystals, amorphous solids, andencapsulated solids, such a

plant extract composition in the form of an emulsion, such a

plant extract composition in an ingestible form selected from capsules,tablets, beadlets, titration packs, powders, drops, lozenges, sprays,syrups, rapidly dissolvable strips and time release capsules, such a

plant extract composition in a non-ingestible form selected from dermalpatches, injectable solutions, drops, suppositories, topical lotions,creams, and sprays, such a

plant extract composition further comprising extracts of Labiatae herbs(including rosemary, sage, oregano, peppermint, basil, spearmint, summersavory), olive extracts, coffee extracts, citrus extracts, tea extracts,tea catechins, catechin, epi-catechin, epi-catechin gallate,epi-gallocatechin gallate, gallic acid, tocopherols, tocotrienols,ascorbic acid and ascorbates (including ascorbyl palmatate), erythorbicacid and erythorbates, glutathione, carnosic acid, carnosol, rosmanol,rosmarinic acid, salviaflaside, flavonoids or flavonoid glucuronides(including quercitin, luteolin, apigenin, or glucuronides of quercitinluteolin, and apigenin and the like), curcumin, tetrahydrocurcumin,hydroxy tyrosol, oleuropein, BHT, BHA, hydroxylamines, propyl gallate,ethoxyquin, Trolox or TBHQ, or mixtures thereof, such a

plant extract composition further comprising extracts of Bixa orellana,Curcuma longa, Daucus carota sativa, Capsicum annuum (other than theinventive plant), Dunaliella salina, Haematacoccus pluvalus,beta-carotene, beta-apo-8-carotenal, the ethyl ester of thebeta-apo-8-carotenoic acid, synthetic colors (FD&C coloring agents),and/or mixtures thereof, such a

plant extract composition which is ingested for human and animal eyehealth and/or to reduce the risk of developing ocular diseases includingcataracts, age-related macular degeneration, Retinitis Pigmentosa, Ushersyndrome, Stargardts, Best's Disease, progressive Cone Dystrophy andretinal degradation, such a

plant extract composition which is ingested for the treatment or theprevention of human or animal diseases including cancer-relateddiseases, cardiovascular diseases, inflammatory disorders and nervoussystem diseases, such a

plant extract composition wherein the cancer-related diseases areselected from breast cancer, gastric cancer and melanoma, such a

plant extract composition wherein the inflammatory disorder is selectedfrom polyarthritis and rheumatoid arthritis, such an

oleoresin composition derived from the plant or regenerable portionthereof, such an

oleoresin composition comprising zeaxanthin, cryptoxanthin, lutein, andother carotenoids, such an

oleoresin composition wherein the oleoresin is substantially free fromterthiophenes, such an

oleoresin composition wherein the oleoresin meets the requirements of 21CFR §73 regulations for spice extractives, such a

presscake derived from the plant, such a

fresh or dried fruit of the plant in either the whole or comminutedform, such

saponified products derived from the plant, such

seasoning and flavoring compositions derived from the plant, comprisingnatural flavors and synthetic flavors, such

pigmenting, flavoring, and/or preserving compositions derived from theplant for animal and human foods, such a

method for the prevention of degenerative or free radical-mediateddiseases including age-related macular degeneration, cataracts,cardiovascular disease and cancer, comprising the step of administeringto a living animal body, including a human, zeaxanthin derived from theplant, or regenerable portion thereof, in a nutritionally effectiveamount for the prevention of such diseases, such a

method for the treatment of degenerative or free radical-mediateddiseases including age-related macular degeneration, cataracts,cardiovascular disease and cancer, comprising the step of administeringto a living animal body, including a human, zeaxanthin derived from theplant, or regenerable portion thereof, in an amount effective to providea therapeutic benefit to the subject suffering from such diseases, sucha

method for reducing the risk of developing ocular disorders selectedfrom cataracts, retinal degeneration, age-related macular degeneration,Stargardts, Best's Disease, progressive Cone Dystrophy, RetinitisPigmentosa, Choroideremia, Ushers Syndrome and Diabetic Retinopathy,comprising the step of administering to a living animal body, includinga human, zeaxanthin derived from the plant, such a

method for reducing the risk of developing free radical-mediateddiseases selected from cancer-related diseases, cardiovascular diseases,inflammatory disorders, nervous system diseases, comprising the step ofadministering to a living animal body, including a human, zeaxanthinderived from the plant, such a

method wherein the cancer-related diseases are selected from breastcancer, gastric cancer and melanoma, such a

method wherein the inflammatory disorders are selected frompolyarthritis and rheumatoid arthritis, such a

method for pigmenting, flavoring, and/or preserving animal and humanfoods comprising the step of incorporating an extract compositionderived from the plant, or regenerable portion thereof, into the animaland human foods, such a

method for pigmenting, flavoring, and/or preserving animal and humanfoods comprising the step of incorporating zeaxanthin derived from theplant, or regenerable portion thereof, into the animal and human foods,such a

method of obtaining a non-esterified zeaxanthin of high puritycomprising:

-   -   (a) contacting ground ripe fruit pods from the plant, or        regenerable portion thereof, of Claim 1 with a solvent for a        time sufficient to extract zeaxanthin from the fruit pods;    -   (b) separating the solvent and extract dissolved therein from        the remaining plant material;    -   (c) desolventizing the extract to obtain a zeaxanthin oleoresin;    -   (d) refluxing the zeaxanthin extract in the dark with butylated        hydroxytoluene, sodium carbonate and potassium hydroxide to        lower the pH; and    -   (e) neutralizing the solution to produce a solution of pure        non-esterified zeaxanthin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an HPLC chromatogram (maxplot 400 nm-600 nm) as set forth inExample 8, of saponified oleoresin derived from the instant Capsicumvarieties. Peak identification is as follows: 1=capsorubin,2=violoxanthin, 3=capsanthin, 4=trans-zeaxanthin, 5=lutein,6=antheraxanthin, 7=9-cis-zeaxanthin, 8=cryptocapsin, 9=α-crytpoxanthin,10=β-cryptoxanthin, 11=ζ-carotene, 12=α-carotene, 13=trans-β-caroteneand 14=cis-β-Carotene. Ratios of zeaxanthin to total carotenoids werecalculated by summing the area counts for all the zeaxanthin isomers anddividing that number by the total area count of all the carotenoidpeaks.

FIG. 2 is an HPLC chromatogram (maxplot 400 nm-600 nm) as set forth inExample 8, of saponified, ground, dried, ripe fruit pod flesh fromCapsicum plants of the present invention. Peak identification is asfollows: 1=capsorubin, 3=capsanthin, 4=trans-zeaxanthin, 5=lutein,6=antheraxanthin, 7=9-cis-zeaxanthin, 9=α-crytpoxanthin,10=β-cryptoxanthin and 13=trans-β-carotene. Ratios of zeaxanthin tototal carotenoids were calculated by summing the area counts for all thezeaxanthin isomers and dividing that number by the total area count ofall the carotenoid peaks.

FIG. 3 shows chromatographic profiles for determining levels ofα-terthienyl as in Example 12. The level of α-terthienyl in a commercialsample of Marigold oleoresin is compared to the α-terthienyl level inthe Capsicum oleoresin of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of this invention, the term zeaxanthin includeszeaxanthin in all of its geometrically isomeric, stereoisomeric andderivatized forms. Lutein is not regarded herein as an isomer ofzeaxanthin. Zeaxanthin geometric isomers include the all-trans form aswell as the various cis isomers such as 9-cis, 13-cis, and 15-cis. Thestereoisomeric forms include 3R,3′R; 3S,3′R; 3R,3′5 and 3S,3′S. Thederivatives of zeaxanthin include both the free hydroxyl form as well asesters with various fatty acids that are typically known to occur in theart. The invention applies to combinations of all these forms ofzeaxanthin. The plants of the instant invention produce predominatelytrans 3R,3′R as a mixture of free hydroxyl compounds and mono- anddi-esters of fatty acids.

The Capsicum genus includes all species and varieties known in the artand that could be developed. These species include but are not limitedto annuum, frutescens, pubescens, chinense.

The instant invention pertains to Capsicum varieties exhibiting ahyper-accumulation of zeaxanthin which are derived through massselection, seed to row evaluation, single plant selection breedingtechniques, or other techniques known in the art. These techniques areused to produce plants having the following characteristics:

-   -   1. The mass of zeaxanthin in the dried ripe fruit pod flesh,        measured as the free or non-esterified diol, is greater than        about 0.4% of the mass of total dried, ripe fruit pod flesh.    -   2. The percentage of zeaxanthin relative to total carotenoids        present in the dried, ripe fruit pod flesh, when measured in the        non-esterified forms, is the dominant carotenoid, defined as the        carotenoid present in the highest concentration, or is greater        than about 50%. The total carotenoids are defined as their free        forms in those cases where esterification is possible. Examples        are capsanthin and cryptoxanthin.    -   3. The mass of zeaxanthin relative to other characteristic        carotenoids present in the dried, ripe fruit pod flesh in highly        pigmented varieties is 4- to 5-fold greater in the instant        orange varieties when compared with commercial red varieties.

Hyper-accumulation is a term open to interpretation. In the context ofthis document, a plant exhibiting a hyper-accumulation of zeaxanthin isone which expresses an amount about twenty-fold higher than the amountexhibited by a standard orange pepper, as defined in Table 1, asproviding 1606 microgram zeaxanthin per 100 gram of raw pepper flesh.Assuming a 92% moisture level in the raw pepper, the weight ofzeaxanthin for this standard pepper would be 0.02% on a dry weightbasis. A pepper exhibiting hyper-accumulation would then contain about0.4% zeaxanthin in the dry, ripe fruit pod flesh.

As used herein the term dried refers to a range of moisture contentstypically observed when paprika is dehydrated. The drying can occur byany means known in the art, including sun drying, oven drying and freezedrying. Moisture contents in dried paprika can range from 1 to 20% byweight, however, typical ranges are between 2 and 10%.

As used herein the flesh of the fruit pod may or may not include thepulp, seeds, stem, placenta, and pericarp.

As used herein the phrase “tissue culture” refers to plant cells orplant parts from which Capsicum plants or plant cultures may begenerated, including plant protoplasts, plant cali, plant clumps, andplant cells that are intact in plants, or part of plants, such as seeds,leaves, stems, pollens, roots, root tips, anthers, ovules, petals,flowers, embryos, fibers and bolls.

Techniques of generating plant tissue culture and regenerating plantsfrom tissue culture are well known in the art. For example, suchtechniques are set forth by Vasil., 1984, Green et al., 1987, Weissbachet al., 1989, Gelvin et al., 1990, Evans et al., 1983, and Klee et al.,1987.

Tissue culture of plant cells or plant parts may be generated from plantprotoplasts, plant cali, plant clumps, and plant cells that are intactin plants, or parts of plants, which when regenerated, produce plants orplant material capable of expressing the morphological and/orphysiological characteristics of the instant Capsicum plants.

Regenerable portions of the Capsicum plants of the present invention,derived from plant cells or protoplasts of a tissue selected from thegroup consisting of embryos, meristems, pollen, leaves, anthers, ovules,roots, root tips, fruit pods, seeds, petals and flowers, fibers andbolls, may be cultured to produce plants or plant material capable ofexpressing all the physiological characteristics of the instant Capsicumvarieties, including a hyper-accumulation of zeaxanthin.

Subcellular constituents of the regenerable cells, comprising nucleicacids, polypeptides and carotenoids, may be isolated from plant cells orprotoplasts of the instant Capsicum plants.

The invention also pertains to the genetic sequences and correspondingamino acid sequences which govern and make possible the hyper-expressionand/or accumulation of zeaxanthin characteristic of the instant Capsicumvarieties.

The nucleic acids of the instant invention may be used to createtransgenic plants or organisms in which the levels of zeaxanthin arepresent at higher than normal levels. To this end, it may be desirableto reduce or eliminate expression of genes encoding carotenoidbiosynthetic enzymes in plants, including capsanthin-capsorubin synthaseor zeaxanthin epoxidase, which downregulation may result in ahyper-accumulation of a carotenoid precursor. Advances in geneticengineering have provided the requisite tools to transform plants tocontain and express foreign genes (Kahl et al., 1995; Hodges, et al.U.S. Pat. No. 5,527,695; Conner, et al., U.S. Pat. No. 6,506,565), aswell as tools to silence the expression of genes in plants throughantisense technologies (Shewmaker, et al. U.S. Pat. No. 5,107,065). Thelimitations of conventional plant breeding may be circumvented by thecreation of transgenic plants genetically engineered to express adesired phenotype (Yin, et al., 2004, and references therein).Therefore, a variety of strategies and molecular techniques can be usedby a skilled artisan to increase the amount of carotenoids in a plant.For example, to increase the level of zeaxanthin in plants, moleculartechniques may be used to transform wild type plants with the instantnucleic acids according to conventional methods in the art to alter theactivity of enzymes of the carotenoid biosynthetic pathway in thoseplants which result is the hyper-accumulation of zeaxanthin.

Breeding

Plants of the instant invention are of the genus Capsicum and are theproduct of a plant breeding program using classical plant breedingmethods of hybridization, single plant selection and progeny rowevaluation.

Seed or Plant Treatment

Alterations in carotenoid biosynthesis, including enhanced production ofzeaxanthin, could lead to changes in the amount or timing of abscisicacid production in the plants of the present invention. It may benecessary to treat the plants or seeds with abscisic acid or abscisicacid precursors or other treatments known in the art at some point intheir development in order to avoid adverse impacts on germination,germination rate or germination timing. Such treatment is known in theart for a wide variety of plants.

Harvesting

Fruit pods produced by plants of the present invention can be harvestedby any means known in the art. The preferred method to harvest theCapsicum species of interest is mechanical. Manual harvesting may alsobe used. The fruit pods can be harvested in their fully hydrated form,providing a pepper for the fresh produce market. The fruit pods can alsobe harvested in a partially desiccated state, after the pods have drieddown on the plant in the field (see examples).

Dehydration

Fresh or partially desiccated pods can be further dried by any meansknown in the art, including sun drying, oven drying, freeze drying andthe like.

Grinding

Desiccated pods can be comminuted or ground by methods known in the art.

Method of Extraction

Zeaxanthin and other carotenoids may be obtained by extraction of thefresh pod flesh, by extraction of the dried fruit, or by extraction of amixture thereof. In some cases, it is preferred to grind the pod fleshinto either a paste or a powder prior to the extraction process. Thegrind profile can be optimized by means known in the art. Extraction canbe done using any of the methods currently known in the art. Theseinclude, but are not limited to extraction with a solvent, or a mixtureof solvents, such as those approved under 21 CFR §173, extraction bymechanical means using a press, such as described in U.S. Pat. No.5,773,075, extraction with sub-critical or supercritical fluids, such assupercritical carbon dioxide in the presence or absence of additionalsolvents or co-solvents, extraction with hydrocarbons, such as ethane,propane or butane, extraction with hydrofluorocarbons, such astetrafluoroethane or with tetrafluoroethane mixed with those organicsolvents approved under 21 CFR §173. Suitable solvents for extractioninclude, but are not limited to n-hexane, cyclohexane, branched hexanes,heptane, branched heptanes, octane, nonane, decane, and otherhydrocarbons. Suitable solvents also include, but are not limited toethyl acetate, tetrahydrofuran, methyl-tert-butyl-ether, ethanol,methanol, acetone, limonene, and other essential oils. As known by thoseskilled in the art, combinations of these solvents can also be utilizedfor the extraction. Those methods that involve the use of a solvent aregenerally followed by a desolventizing process, including, but notlimited to distillation, vacuum distillation, steam distillation,evaporation, steam stripping, nitrogen stripping, membranepervaporation, or molecular distillation.

Further Processing

Whole fresh pods, dried pods, ground pods, or extracts of pods of theinstant Capsicum varieties can be heated to convert all or a portion ofthe naturally occurring all-trans zeaxanthin into cis forms. In asimilar manner, whole fresh pods, dried pods, ground pods, or extractsof pods of the instant Capsicum varieties can be irradiated with lightof wavelength sufficient to convert all or a portion of the naturallyoccurring all-trans zeaxanthin into similar or additional cis forms.Alternatively, the cis forms of zeaxanthin can be converted back intotrans forms by refluxing in ethanol (Khachik et al. 1992), Othercarotenoids such as beta carotene and beta-apo-8-carotenal have alsobeen shown to convert from cis to the all trans form in high yield byheating in petroleum ether or water followed by crystallization (Isier,et al. 1956; U.S. Pat. No. 3,989,757 and references therein). Otherchemical means known in the art may be used to interconvert cis andtrans isomers.

Ground, dried, pod flesh of the Capsicum of the present invention can betreated with caustic solution or with enzymes to saponify or hydrolyzethe zeaxanthin and other carotenoids present in esterified form,together with any other hydrolysable material [see U.S. Pat. No.5,648,564]. The free zeaxanthin and other carotenoids can be separatedfrom the hydrolysate by means known in the art, including dissolving thefree zeaxanthin and other carotenoids in a suitable solvent, filteringor otherwise separating insoluble components from thesolvent/zeaxanthin/other carotenoid mixture, and separation of thesolvent from the zeaxanthin/other carotenoid mixture.

Alternatively, the ground material may be transesterified, using methodsknown in the art, reacting fatty acids with the zeaxanthin (and otherxanthophylls) in order to make a preferred esterified form of zeaxanthinand other xanthophylls.

Further Forms or Formulations of Zeaxanthin

This invention pertains to forms and formulations of zeaxanthin derivedfrom Capsicum varieties, those plants exhibiting a percent of zeaxanthinrelative to total carotenoids which is greater than 50% in the dried,ripe fruit pod flesh, when measured in non-esterified forms. Such formsand formulations are designed and intended for consumption by humans andanimals as nutritional supplements, as food colorants or additives, forthe fortification of human food or animal feeds, or as ingredients incosmetic, personal care or pharmaceutical applications.

Fresh Pepper

The plant product may take the form of the fresh fruit pods harvestedfrom a Capsicum plant, in whole, comminuted, pureed, macerated orexpressed juice form.

Dehydrated Pepper

The plant product may take the form of the dehydrated, dried, ordesiccated fruit pods harvested from a Capsicum plant, in whole,comminuted or ground form. These dehydrated pepper products could reachthe consumer as a pepper powder, seasoning, or as a seasoning in foodsor beverages.

Oleoresin

The plant product may take the form of an oleoresin or extract preparedfrom fruit pods harvested from a Capsicum plant, prepared by any meansknown in the art.

Press Cake

The plant product may take the form of press cake (press solids)produced as a by-product in the press extraction of oleoresin from fruitpods of Capsicum plants, prepared by, but not limited to, the method ofU.S. Pat. No. 5,773,075. The press cake may be used for animal or humannutrition.

Refined Oleoresin

The plant product may take the form of an oleoresin or extract preparedfrom fruit pods harvested from a Capsicum plant, which has been furtherprocessed by any suitable techniques for processing botanical extractsknown in the art, including, but not limited to centrifugation,decanting, precipitation (through seeding or through addition of othersubstances that facilitate precipitation), filtration, crystallizationor recrystallization, saponification, chromatography, membraneprocessing or zone refining, to produce a material with a higherconcentration of zeaxanthin than is present in the initially extractedoleoresin form. The zeaxanthin may be present in these materials inesterified or non-esterified form.

The oleoresin derived from a Capsicum plant of the instant invention maybe refined by the process described by U.S. Pat. No. 6,504,067, theprocess of which is hereby incorporated by reference. The processincludes: 1) refining the plant extract or oleoresin by treatment with adiluted aqueous alkaline solution which forms a first oleoresin phaseand a first aqueous phase containing impurities, 2) treating the firstoleoresin phase with diluted aqueous organic or inorganic acid, 3)forming a second oleoresin phase and a second aqueous phase containingimpurities, and 4) separating the second aqueous phase containingimpurities from the second oleoresin phase to obtain the refinedcarotenoids.

Isolated and Refined Zeaxanthin

Extracts of Capsicum plant pod flesh can be further processed to providezeaxanthin in different forms and purities. Further processing methodsinclude, but are not limited to, centrifugation, decanting,precipitation (through seeding or through addition of other substancesthat facilitate precipitation), filtration, crystallization orrecrystallization, saponification, chromatography, membrane processingor zone refining. The zeaxanthin may be present in these materials inesterified or non-esterified form.

For example, U.S. Pat. No. 5,648,564 discloses a process for forming,isolating and purifying xanthophyll crystals, preferably lutein frommarigold flower petals, zeaxanthin from wolf berries or capsanthin andcapsorubin from red pepper. A xanthophyll diester-containing plantextract is saponified in a composition of propylene glycol and aqueousalkali to form xanthophyll crystals. Crystallization is achieved withoutthe use of added organic solvents. The crystals are isolated andpurified. The substantially pure xanthophyll crystals so obtained aresuitable for human consumption and can be used as a nutritionalsupplement and as an additive in food.

The crystallization process can be used for the purification ofxanthophylls from a saponified extract. U.S. Pat. No. 6,329,557describes a process comprising the steps of dispersing the saponifiedextract in water to form a dispersion, mixing the dispersion underconditions such that a portion of any water-soluble compounds dissolvesin the water to form an aqueous phase and a residue that is not solublein water, separating the aqueous phase from the residue, contacting theresidue with a non-polar solvent under conditions such that a portion ofany lipid-soluble compounds dissolves in the non-polar solvent and aportion of the xanthophylls precipitates from the non-polar solvent toform a precipitate, separating the non-polar solvent from theprecipitate, washing the precipitate with a polar solvent such that atleast a portion of any remaining chlorophylls dissolves in the polarsolvent, and separating the polar solvent from the precipitate to yielda product comprising the xanthophylls at a desired level of purity.

Purified Zeaxanthin in Non-Esterified Form

Zeaxanthin esters from Capsicum plants may be saponified by a number ofmethods described in the art, and they include, but are not limitedto: 1) saponification in water with acid or base, 2) saponification inmethanol or isopropyl alcohol with acid or base, 3) saponification inpropylene glycol with acid or base, and 4) saponification using enzymes.

Oleoresin from the extraction of zeaxanthin from Capsicum plants may besaponified in order to generate free zeaxanthin. This free, all-trans3R,3′R isomer of zeaxanthin can be crystallized to obtain a purifiedform. Thus, the free, all-trans 3R,3′R isomer of zeaxanthin may beformulated in ways similar to those used for all-trans beta-carotene. Aspecific method for producing crystals of lutein and zeaxanthin isdescribed in U.S. Pat. No. 5,648,564.

There are advantages and disadvantages associated with xanthophylls intheir esterified or free forms. U.S. Pat. No. 6,689,400 discloses thatfree lutein is especially vulnerable to chemical and biologicaldeterioration with respect the esterified form. On the other hand, it isdisclosed in U.S. Pat. No. 5,997,922 that the free form of xanthophyllsis more readily absorbed by chickens for egg and flesh pigmentation. Thediester form of lutein appears to be more bioavailable in humans thanthe non-esterified form (Bowen et al., 2002). Breithaupt et al., 2004observed enhanced bioavailability of 3R,3R′ zeaxanthin dipalmitatecompared with the non-esterified form in humans.

Purified Zeaxanthin in Re-Esterified Form

Short chain organic acids may be reacted with free zeaxanthin to produceshort chain organic acid mono- or diesters of zeaxanthin. These shortchain organic acids may be obtained by reacting organic anhydrides witha zeaxanthin extract, as described in U.S. Pat. No. 5,959,138. Theorganic anhydrides that may be used include but are not limited toacetic anhydride, propionic anhydride, and combinations thereof. Otheresters may be formed by esterification processes involving othercarboxylic acids, their anhydrides or esters. Additional zeaxanthinesters can be produced using transesterification reactions whereinzeaxanthin esters are treated with carboxylic acids and an acid, base orenzyme catalyst.

In the case of modified ester forms of zeaxanthin, the carboxylic acidmoieties can consist of short chains (C₁ to C₄), medium chains (C₅ toC₁₂), or longer chains (C₁₃-C₃₀). The carboxylic acid moieties can besaturated, unsaturated or polyunsaturated. The carboxylic acid moietiescan have linear or branched structures.

Isomers of Zeaxanthin

The zeaxanthin present in Capsicum plants consist primarily of the3R,3′R stereoisomer. The pigments found in the fresh fruit of Capsicumplants are overwhelmingly in the all-trans configuration. Transzeaxanthin can be converted in whole or in part into various cis formsby methods known in the art (Khachik, et al., 1992; Updike et al.,2003).

Oleoresin or Purified Forms of Zeaxanthin Dispersed in Oils, Fats,Emulsifiers or Stabilizers, or Combinations Thereof.

Zeaxanthin derived from Capsicum plants can be formulated withhuman-edible or animal-edible ingredients to facilitate its use asnutritional or feed supplements, food or feed colorants, or food or feedadditives. Paprika oleoresin or more highly refined forms of zeaxanthincan be standardized in regard to coloring power by the addition ofvegetable oils, such as corn oil, soybean oil, canola oil, peanut oil,sunflower oil, safflower oil, olive oil, cottonseed oil, palm oil,coconut oil, medium chain triglycerides, triacetin, hydrogenatedvegetable oils, animal fats, such as lard tallow and poultry fat, fishoil, whale oil, algal oil and the like. Paprika oleoresin or more highlyrefined forms of zeaxanthin can be combined with food grade additives,including emulsifiers, such as lecithin, hydroxylated lecithin,monoglycerides, diglycerides, sorbitan esters, such as Polysorbate-80,sucrose esters, polyglycerol esters, tartaric acid esters of mono anddiglycerides, and the like. Specifically, zeaxanthin can be made into ahomogeneous liquid condimental composition useful in flavoring orcoloring foods and beverages and which is dispersible in both oil andwater, comprising: (1) hydroxylated lecithin, (2) tartaric acid estersof mono and diglycerides, and (3) one or more condiments selected fromedible flavorings, edible coloring agents, one of which must bezeaxanthin derived from the present invention, the ratio by weight of(1) plus (2) to (3) being at least 1:4. Paprika oleoresin or more highlyrefined forms of zeaxanthin formulated with food-grade emulsifiers areparticularly useful in beverage applications and emulsion-based foods.

Paprika oleoresin or more highly refined forms of zeaxanthin may becombined with natural and synthetic antioxidants known in the art. Theseinclude, but are not limited to: extracts of Labiatae herbs (includingrosemary, sage, oregano, peppermint, basil, spearmint, summer savory),olive extracts, coffee extracts, citrus extracts, tea extracts, teacatechins, catechin, epi-catechin, epi-catechin gallate,epi-gallocatechin gallate, gallic acid, tocopherols, tocotrienols,ascorbic acid and ascorbates (including ascorbyl palmatate), erythorbicacid and erythorbates, glutathione, carnosic acid, carnosol, rosmanol,rosmarinic acid, salviaflaside, flavonoids or flavonoid glucuronides(including quercitin, luteolin, apigenin, or glucuronides of quercitinluteolin, and apigenin and the like), curcumin, tetrahydrocurcumin,hydroxy tyrosol, oleuropein, BHT, BHA, hydroxylamines, propyl gallate,ethoxyquin, Trolox or TBHQ, or mixtures thereof. Stabilization ofstandard paprika oleoresins with tetrahydrocurcuminoids is described inU.S. Pat. No. 6,689,400.

Oleoresin or Purified Forms of Zeaxanthin in Combination with OtherCarotenoids, Pigments, or Food Colors, or Combinations Thereof.

Paprika oleoresin or more highly refined forms of zeaxanthin can becombined with one or more other natural and/or synthetic carotenoids toprovide mixed carotenoid compositions useful as nutritional or feedsupplements, food or feed colorants, or food or feed additives. Examplesof other natural and/or synthetic carotenoids that can be combinedinclude, but are not limited to: carrot extract, synthetic betacarotene, tomato extract, synthetic lycopene, marigold extract,synthetic lutein, annatto extract, bixin, norbixin,beta-apo-8-carotenal, canthaxanthin, astaxanthin, lutein, algalcarotenoids, fungal carotenoids, cryptoxanthin, alpha-zeacarotene,beta-zeacarotene, and the like.

Paprika oleoresin or more highly refined forms of zeaxanthin can becombined with other natural or synthetic approved food colorants tocreate compositions useful for providing a range of color hues for foodor feed applications. Natural or synthetic colorants that can becombined include, but are not limited to: turmeric extract, purplecarrot extract, anthocyanins, grape skin extract, beet extract, cabbageextracts, elderberry extracts, caramel, betalins, chlorophyll andapproved FD&C food colorants.

Oleoresin or Purified Forms of Zeaxanthin Dispersed on Solids Suitablefor Nutritional Supplement, Food, Beverage, Cosmetic or PharmaceuticalApplications.

Zeaxanthin in an oleoresin or more highly purified form may be dispersedonto a wide variety of solid carriers suitable for use in a wide varietyof applications. The carriers can include salt, dextrose, maltodextrin,lactose, lignin, flour, talc, titanium dioxide, pharmaceutical andcosmetic excipients or other solid substrates or combinations thereof.Stable cold-water dispersible preparations of carotenoids produced fromzeaxanthin obtained from Capsicum plants comprise carotenoids and awater-soluble or water-dispersible lignin derivative used in place ofgelatin from warm-blooded animals (U.S. Pat. No. 5,668,183). The ligninderivatives for the preparations can contain a single lignin or amixture of several lignin derivatives. Sodium, calcium, and ammoniumlignosulphonate are especially preferred.

In addition, cold water dispersible forms can be made using starches,gums, or other methods known in the art.

Oleoresin or Purified Forms of Zeaxanthin in the Form of BeadletsSuitable for Nutritional Supplement, Food, Beverage, Cosmetic orPharmaceutical Applications.

Beadlets, or microcapsules may be made that contain zeaxanthin, in anyof its forms. Typical carotenoid concentrations range between 1 and 50percent by weight. The microcapsules release the encapsulatedcarotenoids during the ingestion process. These microcapsules may alsobe suitable for use in human or animal foods, multivitamins, dietarysupplements, and personal care products. They may also be used intableting and capsules.

Zeaxanthin used to make beadlets may be either in the form of acrystalline powder or oil dispersion. Starting with a crystalline powderis preferable in some cases so that beadlets containing higherconcentrations of zeaxanthin may be obtained.

Either chemical or physical methods of microencapsulation known in theart can be used to encapsulate zeaxanthin from the instant invention.Chemical methods of microencapsulation include, but are not limited tothose involving: phase separation, solvent evaporation, solventextraction, interfacial polymerization, simple and complex coacervation,in-situ polymerization, liposome technology, nanoencapsulation, sol-gelmethods, vapor-phase deposition, entrapment/matrix encapsulation,macroemulsion, dispersion polymerization, desolvation, and gelation.Physical methods for encapsulation include but are not limited to spraydrying, spray cooling, rotary disk atomization, fluid bed coating,stationary nozzle coextrusion, centrifugal head coextrusion, submergednozzle coextrusion, pan coating, vibrating nozzle, extrusion, prilling,and annular jet methods.

In one form of the process, the crystalline powder is added to afluidized bed dryer and the flow of heat and gas started. A liquidcoating material is sprayed onto the solid to the desired formulation.The liquid coating material may comprise, but is not limited to anaqueous solution of a sugar, or sorbitol, a starch or maltodextrin, andoptionally a coating protein such as gelatin. Details of the process aredisclosed in U.S. Pat. No. 6,663,900. Zeaxanthin-rich oil dispersions orhigher-purity crystalline forms of zeaxanthin can be encapsulated in amatrix such as that described in U.S. Pat. No. 5,786,017, U.S. Pat. No.5,506,353 and U.S. Pat. No. 6,607,771.

Zeaxanthin derived from the instant invention may be combined withmatrix materials known in the art in the process of encapsulation. Ingeneral, hydrocolloids, carbohydrates, and other compounds may be used.Hydrocolloids include, but are not limited to gelatin, milk proteins,vegetable proteins, animal proteins, gums, and modified starches. Thecarbohydrates include, but are not limited to sucrose, glucose, anddextrins. Other components include antioxidants, emulsifiers,stabilizers, and weighting agents.

Zeaxanthin derived from the instant invention may be combined withsurface ingredients known in the art in the process of encapsulation.These agents include, but are not limited to proteins, carbohydrates,silicates, polysaccharides, polyhydric alcohol, waxes, fats, natural andsynthetic polymers, resins, gelatin, polyvinyl alcohol, maltodextrin,methyl cellulose, polyvinyl pyrrolidone, and polyoxymethylene urea.

Zeaxanthin derived from the instant invention may be placed intomicrocapsules for improved heat, chemical, light, and oxidativestability; for better shelf life; and for improved color, odor,taste-masking, and handling. Zeaxanthin derived from the instantinvention in encapsulated form may improve bioavailability; modifysolubility; and offer controlled, sustained, delayed, pulsatile, pHinduced, or targeted release.

Oleoresin or Purified Forms of Zeaxanthin in the Form of Emulsions.

Emulsions of zeaxanthin derived from Capsicum plants can be formed bytechniques well known in the art. Emulsions of zeaxanthin may beprepared for use in aqueous systems of food, beverage, cosmetic,pharmaceutical and personal care products. The combination of asurfactant, zeaxanthin, optionally in an oil carrier, and, optionally,an anti-foaming agent are used to produce an aqueous emulsion. Theemulsion may be dried to form a powder that is readily dispersible in anaqueous medium. The zeaxanthin used in emulsions can take the form of acrude oleoresin or a more highly purified form of zeaxanthin that iseither free zeaxanthin or zeaxanthin in an esterified form.

Alternatively, an emulsion of zeaxanthin may be prepared following U.S.Pat. No. 6,296,877 as: 1) preparing a homogenous solution of zeaxanthinoptionally with an emulsifier and optionally an edible oil in a watermiscible organic solvent, 2) mixing this solution with an aqueoussolution of a mixture of protective colloids, and optionally 3)preparing a water-dispersible dry powder by freeing the resultingdispersion from the solvent and water and drying it.

Oleoresin or Purified Forms of Zeaxanthin in the Form of Spray-Dried orEncapsulated Powders Suitable for Nutritional Supplement, Food,Beverage, Cosmetic, Personal Care or Pharmaceutical Applications.

Zeaxanthin derived from Capsicum plants is suitable for processing intopowdered forms. Emulsions of zeaxanthin oleoresins or dispersions ofzeaxanthin crystals or pulverized forms of zeaxanthin can be spray driedusing technology known in the art. Commonly, an oil dispersion orsolution of zeaxanthin is mixed with water and a polymeric material,such as gelatin, vegetable gum, modified starch, dextrin, or non-gellingproteins. An emulsifier is added and the mixture is homogenized. Theresulting emulsion is atomized and introduced into a heated column ofair in a drying chamber, and free-flowing powders are produced as thewater is evaporated. An example of this kind of method is described inU.S. Pat. No. 6,635,293. Solid zeaxanthin forms can be encapsulated byany of a variety of techniques known in the art, including fluid bedagglomeration or coacervation.

Another example of producing a dry product is disclosed in U.S. Pat. No.3,998,753. Carotenoid powder compositions derived from Capsicum plantsthat are dispersible in aqueous solutions and that form optically clearaqueous compositions that color these aqueous solutions to a desireduniform color can be made as follows. First, a solution of a carotenoidin a volatile organic solvent capable of solublizing carotenoids isformed and emulsified with an aqueous solution containing a food-gradesurfactant using high-speed mixing. The volatile solvent is then removedfrom the resulting emulsion by heating the emulsion while maintainingthe high speed mixing with high shear until the solvent is completelyremoved. The emulsion can then be used as is or dried to yieldcarotenoid-containing powder compositions.

Oleoresin or Purified Forms of Zeaxanthin Encapsulated in a Manner toAllow Incorporation into Nutritional Supplement, Food, Beverage,Cosmetic or Pharmaceutical Applications, without Altering the Color ofthe Nutritional Supplement, Food, Beverage, Cosmetic or Pharmaceutical.

Zeaxanthin particles or particles containing zeaxanthin can be coatedwith opaque materials that effectively hide the color of the pigment.This is a useful technique if it is desired to add zeaxanthin to aproduct without changing the product's color.

Applications of Zeaxanthin Formulations

Zeaxanthin-Rich Food

Fruit pods from the Capsicum varieties of the present inventionexhibiting a hyper-accumulation of carotenoid pigment may be useddirectly as a human food and the juice expressed from them used as adrink. The Capsicum fruit pods may be consumed in the fresh or driedstate. These forms may be ground, chopped, or liquefied for use alone orin combination with any other food, sauce, or beverage. The material maybe used as a component in a seasoning or condiment.

Food Color Applications

Naturally derived carotenoids have attained considerable importance ascoloring agents, and their importance has increased due to governmentregulations withdrawing or limiting the use of certain previouslycertified coloring agents. The pigment in the Capsicum varieties of thepresent invention is acceptable as a food coloring agent in the UnitedStates under FDA regulation (21 CFR §73.340). Capsicum-derived pigmentis the only known source of zeaxanthin that can be used as a foodcolorant under current food regulations. There is considerable interestin the availability of light-stable yellow colorants in the foodindustry to replace the light-unstable curcumin pigments derived fromturmeric (Curcuma longa). Zeaxanthin products derived from the Capsicumvarieties can be added to various foods and beverages for humanconsumption as a nutritional food-coloring agent to provide a bright,natural yellow appearance with high light stability relative toturmeric.

Fat containing foods including, but not limited to, butter, margarines,vegetable oils, chocolate, baked goods such as cakes, breads, bagels,crackers, pizza dough, pancakes and waffles and mixes for these,fillings, peanut butter, salad dressings, processed cheese, processedmeats, seasoning blends and sauces can all be colored by the addition ofthe inventive zeaxanthin oleoresins or purified forms of the inventivecompositions dispersed in oils, fats and emulsifiers. Said formulationsof the instant Capsicum varieties containing high concentrations ofzeaxanthin may also contain natural and synthetic antioxidants known inthe art.

Dry food products including, but not limited to, bakery mixes includingbread mix, bagel mix, cake mix, pizza dough mix, cereals, dry soups,seasoning blends, tomato powder, cereals, macaroni, pasta flour,nutritional and energy bars can all be colored by direct addition ofpowdered formulations of zeaxanthin. These powder formulations may havebeen produced by spray-drying, encapsulation or dry dispersing theoleoresin or refined oleoresin onto a dry carrier. Said formulations mayalso contain natural and synthetic antioxidants known in the art.

Seasoning formulations comprising the inventive zeaxanthin compositionsin combinations with flavoring and/or preserving agents arecontemplated. Flavoring agents include but are not limited to spice andherb extractives, synthetic flavorings, essential oils, fixed oils, andthe like. Preserving agents include but are not limited to natural andsynthetic antioxidants known in the art, some of which are listed above.These compositions can optionally include carriers and/or excipientsincluding but not limited to vegetable oils, ethanol, water, propyleneglycol, glycerin, benzyl alcohol, monoglycerides, diglycerides, andother emulsifiers or combinations thereof as described above or known inthe art.

Coloring formulations comprising the inventive zeaxanthin compositionsin combinations with coloring and/or preserving agents are contemplated.Coloring agents include but are not limited to extracts of Bixaorellana, Curcuma longa, Daucus carota sativa, Capsicum annuum (otherthan the instant plant), Dunaliella salina, Haematacoccus pluvalus,beta-carotene, beta-apo-8-carotenal, the ethyl ester of thebeta-apo-8-carotenoic acid, synthetic colors (FD&C coloring agents), andthe like. Preserving agents include but are not limited to natural andsynthetic antioxidants known in the art, some of which are listed above.These compositions can optionally include carriers and/or excipientsincluding but not limited to vegetable oils, ethanol, water, propyleneglycol, glycerin, benzyl alcohol, monoglycerides, diglycerides, andother emulsifiers or combinations thereof as described above or known inthe art.

Aqueous-based foods including, but not limited to, sauces, includingtomato sauce, steak sauce, and pizza sauce, gravies, soups, gelatins,puddings, eggnog, ketchup, pickles, salad dressings, egg yolks, meatmarinades, dairy products, such as milk, yogurt, and ice cream, can allbe colored by direct addition of water soluble powdered formulations ofzeaxanthin. They may alternately be colored by the addition ofzeaxanthin oleoresin, which has been admixed with emulsifiers such asmono-glycerides, tartaric acid esters of triglycerides, lecithins,polysorbates sucrose fatty acid esters or hydroxylated lecithins, ormixtures thereof, to form a water dispersible resin. Said formulationsmay also contain natural and synthetic antioxidants known in the art.

Beverages, including but not limited to, nutritional drinks, sodas,milk, beer, alcoholic beverages, fruit juices (including, but notlimited to orange juice, apple juice, grape juice, cranberry juice,tomato juice, guava juice, mango juice, cantaloupe juice, carrot juice,and grapefruit juice), dairy beverages, soy beverages, infant formulas,adult formulas (including Ensure®—a registered trademark of AbbottLaboratories Corporation) and their concentrates can be colored orfortified with a zeaxanthin product in the form of water dispersiblepowders produced by spray drying or encapsulation. They may alternatelybe colored with aqueous emulsion or emulsified resin forms containingthe zeaxanthin product. Said formulation may also contain natural andsynthetic antioxidants known in the art.

There is a substantial body of published articles and patents relatingto the formulation and use of beta-carotene and other carotenoids asfood colorings, nutritional additives, feed supplement, cosmeticadditives, personal care additives and pharmaceutical additives. Suchpublications include, for example, U.S. Pat. No. 4,522,743, U.S. Pat.No. 5,180,747, U.S. Pat. No. 5,350,773 and U.S. Pat. No. 5,356,636. Dueto the similarities in the chemical and physical properties ofzeaxanthin and beta carotene, or other carotenoids, any technique,additive, stabilizer, or other method for adding beta-carotene or othercarotenoids to any type of food, cosmetic, feed, pharmaceutical,personal care or nutritional use is also likely to be directlyapplicable to the zeaxanthin compositions derived from the instantCapsicum varieties.

Cosmetics

Zeaxanthin derived from the instant Capsicum plants may be used invarious types of cosmetic applications. It can be applied topically ortaken internally for sun protection and as an antioxidant. Zeaxanthincan be used in lip applications such as lip balms, lipsticks, lipliners, lip moisturizers, and the like. Zeaxanthin may be used incosmetic applications that include foundations, makeup, blushes, tanningcreams, and the like. It can also be used in topical products that areapplied to the skin for protection from the effects of radiation, suchas that from the sun. These products include tanning lotions, tanningaccelerators, tanning moisturizers, and the like. An example of usingzeaxanthin in cosmetic applications is U.S. Pat. No. 6,110,478, whichdiscloses a composition for cosmetic purposes which is a regulator ofcutaneous pigmentation and is adapted both to administration by the oralroute and to application on the skin.

Animal Feed

Zeaxanthin derived from the instant Capsicum varieties may beadministered to animals in order to pigment their flesh, skin or theireggs, or to serve as a nutritional supplement.

Pigmentation of Fish and Crustaceans

Zeaxanthin derived from the instant Capsicum varieties may beadministered to fish or crustaceans in order to pigment their flesh. Forexample, salmon can be fed the inventive product in order to create aflesh tone that is appealing to consumers. Likewise the flesh ofcrustaceans such as shrimp, prawns, lobsters, and crawfish can bepigmented to a more desirable product color.

Broiler Skin and Egg Pigmentation

The color of poultry broiler skin and of egg yolk is widely known as animportant quality attribute. Each region of the world has establishedits own particular specification for this parameter. Thus, the optimumpigmentation of broiler skin and egg yolk depends on cultural traditionsand preferences. Traditionally, poultry keepers have been incorporatingred and yellow pigments (natural or synthetic) into the bird's feed.

Synthetic canthaxanthin has been used for decades as a pigment toprovide a yellow-orange color to poultry broiler skin, and to provideintense orange and even rose hues to egg yolk (U.S. Pat. No. 5,997,922).

U.S. Pat. No. 3,539,686 discloses that it is possible to obtain a widerange of tones going from yellow to red hues in broiler skin and eggyolk, by using blends of xanthophylls or zeaxanthin with one or morepigments such as canthaxanthin, beta-apo-8-carotenal, ethyl ester of thebeta-apo-8-carotenoic acid, and extracts from paprika and red peppers.U.S. Pat. No. 3,539,686 discloses that it is a requirement to use a redpigment in order to obtain more intense orange or reddish hues, ascompared with the hues obtained if only yellow xanthophylls were used,because of the synergistic effect obtained when both pigments are used.

A great amount of research has been performed to determine the differentproportions of yellow xanthophylls and red pigments in order to obtainspecific hues in broiler skin and in egg yolk (see U.S. Pat. No.5,997,922). It has been demonstrated that zeaxanthin provides moreefficient pigmentation than lutein, by imparting an orange hue to thebroiler skin.

Traditional sources of yellow xanthophylls are alfalfa, yellow corngluten, and marigold meal concentrates, wherein it has been demonstratedthat the saponified natural pigment has a better bioavailability thanthe non-saponified pigment in poultry (U.S. Pat. No. 5,997,922).

A preparation of a saponified marigold extraction with a high content ofzeaxanthin, called Hi-Gold® (Organica, S.A. de C.V.) obtained by aprocess for the isomerization of lutein contained in the extract, as isdescribed in U.S. Pat. No. 5,523,494. This patent describes theapplication of Hi-Gold® for broiler skin and egg yolk pigmentationpurposes eliminating the use of red pigments. Furthermore, it has beendemonstrated that by using Hi-Gold®, deeper hues are obtained in broilerskin and egg yolk than those obtained when only the traditionally yellowpigments are used alone.

U.S. Pat. No. 5,997,922 discloses a method for orange tone pigmentationof broiler skin and egg yolk, comprising: dosing about 8 to 55 ppm ofsaponified xanthophylls having a zeaxanthin content of about 20 to 80%in the feed, beverage, or broth, of broilers and laying hens, in theabsence of natural or synthetic red pigments.

Zeaxanthin derived from the instant Capsicum varieties may be used forpigmentation of broiler skin and egg yolk.

Animal Nutrition

Zeaxanthin derived from the instant Capsicum varieties may beadministered to pets, livestock, and other animals as a dietarysupplement as well as to prevent diseases such as cataracts, AMD andother degenerative diseases. The inventive zeaxanthin may be used as adietary supplement for dogs, cats, cattle, horses, sheep, fish, goats,rabbits, chickens, turkeys, and other animals. Zeaxanthin may beadministered to these animals in a wide variety of forms known in theart. This includes, but is not limited to tablets, dips, food, treats,and pellets. In aquaculture, zeaxanthin may be used to impart a desiredcolor to the body and/or flesh.

Human Nutrition

Zeaxanthin is disclosed to be effective in the treatment of a variety ofeye diseases (U.S. Pat. No. 5,854,015). Stereoisomeric forms ofzeaxanthin and their use in the treatment and prevention of AMD andother eye disorders is disclosed in U.S. Pat. No. 6,329,432. Zeaxanthinderived from the instant Capsicum varieties confers advantages over thezeaxanthin compositions of the prior art for the reasons identifiedherein and are summarized again as follows:

-   -   1. The zeaxanthin compositions derived from the instant Capsicum        varieties do not contain nor were ever contacted with phototoxic        or contact dermatitis sensitizing agents.    -   2. The zeaxanthin compositions derived from the instant Capsicum        varieties can be used as a natural food colorant under existing        food regulations.    -   3. The zeaxanthin compositions derived from the instant Capsicum        varieties are GRAS and can be used as a food additive.    -   4. The zeaxanthin compositions derived from the instant Capsicum        varieties are natural products.    -   5. Fruits of Capsicum are a common food source.

All patents cited in this application are herein incorporated byreference.

EXPERIMENTAL PART

The subject matter of the instant invention will be better understood inconnection with the following examples, which are intended as anillustration of and not a limitation upon the scope of the invention. Itwill be apparent to those skilled in the art that the described examplesare merely representative in nature.

Example 1 Development of a Capsicum Plant Exhibiting High Concentrationsof Zeaxanthin

The instant Capsicum plants exhibiting high concentrations of zeaxanthinin the ripe fruit pod flesh were developed using classical plantbreeding methods, and methods known to those skilled in the art, whichare understood in the art to encompass inbreeding and out-crossing,using a commercial Capsicum annuum variety NM plant type as a source ofplant breeding material. The instant varieties resulted from thedevelopment of plant varieties which exhibit a high concentration ofcarotenoid pigments in the fruit pods. High carotenoid concentration andappropriate plant habit are desired for commercially adapted varieties.

During breeding and selection, Capsicum plants of the instant invention,exhibiting high concentrations of zeaxanthin in the ripe fruit podflesh, may easily be distinguished from the wild type red-fruitedvariety by its distinguishing orange color. Capsicum plants of theinstant invention exhibit a sufficiently different appearance to allowone skilled in the art to distinguish it from other Capsicum annuumvarieties.

Screening for Capsicum varieties exhibiting high concentrations ofzeaxanthin in the ripe fruit pod flesh, is routinely carried out byfirst selecting for the desired plant morphology and subsequent analysisof carotenoid composition in the fruit. Capsicum plants exhibiting highzeaxanthin concentrations in the mature fruits can predictably be bredby inbreeding the commercially grown NM variety, NM 1441, deposited atthe American Type Culture Collection (ATCC) under the depositdesignation PTA-10729, as a parental strain (NM 1441×NM 1441). As theplant habit and color of ripe fruit pods of the instant Capsicum varietyexhibit a phenotype which is markedly different from the parentalvariety, screening for plants exhibiting the desired phenotype is easilycarried out by visual inspection of the plant rows. Obtaining a Capsicumplant with the desired zeaxanthin composition by using the breedingmethods described herein is a relatively rare, but a repeatable event.For example, 5 Capsicum plants exhibiting high concentrations ofzeaxanthin in the ripe fruit pod flesh were obtained after evaluation ofabout 102,000 plants in a test plot. Routine screening of this number ofplants for the desired phenotype was easily carried out by rowevaluation for the readily identifiable visual differences in the maturefruit of the instant variety (orange fruit) compared to the parental, orwild-type variety (red fruit) and the morphological differences in planthabit. Following selection, the zeaxanthin content in the ripe fruit podflesh was confirmed by HPLC analysis.

Using similar methods, other plants of the Capsicum genus can be used todevelop varieties that hyper-accumulate zeaxanthin.

Example 2 Describing Small-Scale Field Production

A small test plot of the Capsicum varieties of the instant invention wasplanted. After 6 months, the crop was treated with a defoliant andallowed to dry in the field. The crop was hand-harvested. The fruit podswere sliced and dehydrated in a commercial continuous gas-fired oven.Two composite samples of the dried fruit pods from production wereanalyzed as described in Examples 6, 8 and 9. The mass percentzeaxanthin relative to total carotenoids was 70.81% and 71.80%. Theweight percent of zeaxanthin in the ripe, dried fruit pods was 0.93% and0.97%.

Example 3 Describing Field Production

A test plot of the instant Capsicum varieties was planted. After 6months, the crop was treated with a defoliant and allowed to dry in thefield. The crop was hand-harvested. The fruit pods were sliced anddehydrated in a commercial continuous gas-fired oven. Seven lots offruit pods were harvested and representative samples of each wereanalyzed as described in Examples 6, 8 and 9. The mass percentzeaxanthin relative to total carotenoids in the seven samples was 57.7%,59.7%, 61.3%, 59.1%, 59.6%, 53.3%, and 60.9%. The mass percent ofzeaxanthin in the dried, ripe fruit pods was 0.76%, 0.81%, 1.0%, 0.96%,0.70%, 0.83%, and 0.87% of the total dried ripe fruit pod flesh,respectively.

Example 4 Extraction of the Instant Capsicum Varieties on a CommercialScale

2894 pounds of the dried, ripe fruit pods of the instant Capsicum plantsfrom Example 2 was ground and solvent-extracted with mixture of hexaneand acetone (65:35) in a continuous basket extractor. The miscella wasdesolventized to less than 25 ppm hexanes and acetone using a vacuumstripper to obtain 194 pounds of a Capsicum oleoresin. The oleoresincontained 6.7% total zeaxanthin, measured as free zeaxanthin after asaponification step (using the methods of Examples 7 and 9).

Example 5 Determination of Zeaxanthin Stereochemistry

The optical isomer present in the oleoresin was determined by analysisin a commercial laboratory. Ground input material was extracted as inExample 4 and saponified on a small scale, along the lines of Example 6.This saponified sample was dissolved in tetrahydrofuran and diluted forcarotenoid analysis on a C30 column using gradient separation. Foroptical isomer analysis, the samples were dissolved in hexane andexamined by normal-phase HPLC for xanthophyll content. For samplescontaining multiple peaks, the zeaxanthin peak was collected frommultiple injections on this system. The combined collection wasre-injected to ensure that only trans-zeaxanthin had been collected. Thecombined collections were concentrated under nitrogen and injected ontoa series of two chiral Chiralcel® OD columns (4.6×250 mm, 5 μm) (DaicelChemical Industries, LTD, Fort Lee, N.J.). The mobile phase used in theseparation was 5% isopropanol in hexane at a flow rate of 0.6 mL/min.The analytes were detected at 450 nm. A neat standard of 3R,3′Rzeaxanthin was injected with each set of samples to verify the retentiontime. For any samples in which the retention time of the zeaxanthin peakdid not match that of 3R, 3′R zeaxanthin, the samples were spiked withthis standard and re-injected to distinguish between retention timeshifts and distinctly different peaks. The sample was found to containonly one optical isomer that was identified as 3R, 3′R zeaxanthin.

Example 6 Saponification Procedure of Ground Capsicum for HPLC Analysis

Ground, ripe, dried fruit pod flesh of the instant Capsicum varieties(1.0 g) was weighed to the nearest tenth of a milligram on an analyticalbalance and was quantitatively transferred to a 125 ml Erlenmeyer flask.The flask was immediately covered with aluminum foil to reduce exposureto light. Butylated hydroxytoluene (0.2 g, Sigma Chemical Company), and1.5 g of sodium carbonate powder (Aldrich Chemical—A.C.S. reagent) wereweighed and added to the Erlenmeyer flask. 50 ml of methanol (FisherScientific—HPLC-grade) and about 0.8 g of potassium hydroxide (VWRIntl.) were added to the Erlenmeyer flask. A stir bar was added to thesolution and a Vigreux distilling column was attached to the top of theErlenmeyer flask. The solution was placed on a hot plate and refluxed onlow heat (˜65° C.) with stirring for 1 hr. Then the solution was takenoff the hot plate and allowed to cool. A total of 1.2 ml of phosphoricacid (Innophos 75% FCC grade; Innophos, Inc., Cranbury, N.J.) was addedto neutralize the solution. The solution was vacuum filtered through aBuchner funnel containing Celite® (Eagle Picher Filtration and Minerals,Reno, Nev.) directly into a 200 ml volumetric flask. All the color wasrinsed out of the Erlenmeyer flask and the Buchner funnel with methanol,combined and brought to a 200 ml total volume with methanol. Afterinverting the flask several times, the solution was poured into a 3 ccsyringe with a 0.45 micron PTFE Acrodisc® (Gelman) filter and injectedinto an amber vial for HPLC analysis.

Example 7 Oleoresin Saponification Procedure for HPLC Analysis

Oleoresin derived from the instant Capsicum varieties (0.03 g) wasweighed to the nearest tenth of a milligram on an analytical balancedirectly into a 125 ml Erlenmeyer flask. The flask was immediatelycovered with aluminum foil to reduce exposure to light. A total of 0.2 gof butylated hydroxytoluene (Sigma Chemical Company) and 1.5 g of sodiumcarbonate powder (Aldrich Chemical—A.C.S. reagent) were weighed andadded to the Erlenmeyer flask. 50 ml of methanol (FisherScientific-HPLC-grade) and about 0.8 g of potassium hydroxide (VWRIntl.) were added to the Erlenmeyer flask. A stir bar was added to thesolution and a Vigreux distilling column was attached to the top of theErlenmeyer flask. The solution was placed on a hot plate and refluxed onlow heat (˜65° C.) with stirring for 1 hr. Then the solution was takenoff the hot plate and allowed to cool. A total of 1.2 ml of phosphoricacid (Innophos 75% FCC grade) was added to neutralize the solution. Thesolution was vacuum filtered through a Buchner funnel containing Celite®(Eagle Picher Filtration and Minerals, Reno, Nev.) directly into a 200ml volumetric flask. All the color was rinsed out of the Erlenmeyerflask and the Buchner funnel with methanol, combined and brought to a200 ml total volume with methanol. After inverting the flask severaltimes, the solution was poured into a 3 cc syringe with an 0.45 micronPTFE Acrodisc® (Gelman) filter and injected into an amber vial for HPLCanalysis.

Example 8 Determination of the Percentage of Zeaxanthin Relative toTotal Carotenoids (Area %) by HPLC

Analyses were performed on a Waters 2695 (Milford, Mass. USA) separationsystem using Empower (Build 1154, Database version 5.00.00.00) softwareinstalled on the data station. The chromatographic separation wasperformed on a reverse-phase column (Waters Symmetry® C18, particle size5 μm, 250 mm×4.6 mm). The eluent was a ternary gradient ofmethanol/water/acetone at 1.0 ml/min. The initial composition of theeluent was methanol-water-acetone (0:25:75, v/v/v). An initial lineargradient was applied for 15 minutes that yielded a composition ofmethanol-water-acetone (20:5:75, v/v/v). This composition was held for15 minutes, followed by another linear gradient for 30 minutes to yielda composition of methanol-water-acetone (25:0:75, v/v/v). Finallyanother linear gradient was applied for 15 minutes yielding acomposition of methanol-water-acetone (0:0:100, v/v/v). This compositionwas held for 5 minutes and returned to initial conditions. Compoundswere detected (maxplot between 400 nm-600 nm) on a Waters 996 photodiodearray detector using an injection volume of 20.0 μl. Literatureretention times and PDA spectra were used to identify some of the peaks(violoxanthin, antheraxanthin, 9-cis-zeaxanthin, cryptocapsin,α-cryptoxanthin, ζ-carotene). Other compounds were identified andcompared with standards from Carotenature (Lupsingen, Switzerland) andare listed as follows: capsorubin, capsanthin, trans-zeaxanthin, lutein,β-cryptoxanthin, α-carotene, trans-β-carotene and cis-β-carotene.Example chromatograms of saponified ground, ripe, dried fruit pods andsaponified oleoresin from the instant Capsicum varieties are included asFIGS. 1 and 2, respectively.

Example 9 Determination of Zeaxanthin Content (Wt %) by HPLC

The analyses were performed on a Waters 2695 (Milford, Mass. USA)separation system using Empower (Build 1154, Database version5.00.00.00) software installed on the data station. The chromatographicseparation was performed on a reverse-phase column (Waters Symmetry®C18, particle size 5 μm, 250 mm×4.6 mm). The eluent was a ternarygradient of methanol/water/acetone at 1.0 ml/min. The initialcomposition of the eluent was methanol-water-acetone (0:25:75, v/v/v).An initial linear gradient was applied for 15 minutes and yielded acomposition of methanol-water-acetone (20:5:75, v/v/v). This compositionwas held for 15 minutes, followed by another linear gradient for 30minutes to yield a composition of methanol-water-acetone (25:0:75,v/v/v). Finally another linear gradient was applied for 15 minutesyielding a composition of methanol-water-acetone (0:0:100, v/v/v). Thiscomposition was held for 5 minutes and then returned to initialconditions. Compounds were detected (maxplot between 400 nm-600 nm) on aWaters 996 photodiode array detector using an injection volume of 20.0μl. Zeaxanthin content was measured in reference to a calibration curvegenerated from a purchased authentic sample. Trans-zeaxanthin obtainedfrom Carotenature (Lupsingen, Switzerland) was dissolved in methanol.This stock solution was used to generate a 5-point external calibrationcurve covering concentrations ranging from 2.0 μg/ml-45.0 μg/ml.9-cis-zeaxanthin was quantified using the trans-zeaxanthin calibrationcurve, assuming a response factor of 1:1. Zeaxanthin contents arereported as a sum of all zeaxanthin isomers.

Example 10 Random Sampling of Production Field

Ten field samples of random individual Capsicum plants of the instantinvention were harvested. The pods were de-seeded and dehydrated in alaboratory dehydrator and were subjected to analysis as described inExamples 6 and 8. The percentage of zeaxanthin relative to totalcarotenoids, measured in non-esterified forms, in each sample is shownbelow.

Sample % Zeaxanthin to total Carotenoids 6203017a 68.99 6203023a 69.846203037a 71.00 6203038a 70.21 6203045a 77.87 6203053a 65.72 6203058a67.83 6203060a 67.30 6203065b 69.59 6203073a 62.96

Example 11 Random Sampling of Production Field

Sixty-three field samples of random individual Capsicum plants of thepresent invention were harvested. The pods were de-seeded and dehydratedin a laboratory dehydrator and were subjected to analysis as describedin Examples 6, 8 and 9. The percentage of zeaxanthin relative to totalcarotenoids, measured in non-esterified forms, in each sample is shownbelow. The mass percent of zeaxanthin, measured as the free diol, forselected samples is also shown. The HPLC method utilized the calibrationcurve described in Examples 8, and 9. The ASTA value and % zeaxanthinbased on ASTA was determined by the method in Example 23.

Calculated mass % Measured % Zeaxanthin Zeaxanthin mass % to total basedon zeaxanthin Sample Carotenoids ASTA Value ASTA Value by HPLC 9004008a73.2 539 1.0 9004013a 68.9 465 0.8 9004027a 70.7 490 0.9 9004061a 67.5414 0.7 9004075a 76.3 454 0.9 9004082a 68.9 383 0.7 9004085a 75.2 5481.1 9004127a 76 381 0.8 9004183a 73.1 426 0.8 9004213a 68.8 511 0.99004214a 75.9 494 1.0 9004219a 74 474 0.9 9004289a 73.4 517 1.0 9004053a71.5 428 0.8 9004098a 73.7 422 0.8 9004139a 72.6 395 0.7 9004189a 74.5349 0.7 9004261a 73.7 456 0.9 9004299a 65.9 430 0.7 0.885 9004343a 74.1344 0.7 0.761 9004395a 73.6 423 0.8 9004435a 73.6 343 0.7 9004492a 61.8448 0.7 0090005a 72.6 584 1.1 1.373 0115001a 71.6 520 1.0 0185002a 70.8523 1.0 0199001a 68.5 514 0.9 0199002a 65.1 561 1.0 0220003a 67.7 4890.9 1.075 0320001a 66.1 506 0.9 0335002a 70.7 524 1.0 0360001a 68.5 5150.9 0435004a 65.5 510 0.9 0.989 8004003a 69.5 546 1.0 9004016a 54.7 3920.6 9004029a 69.8 543 1.0 9004058a 60.2 398 0.6 9004068a 67.2 481 0.89004077a 71.6 415 0.8 9004087a 69.6 318 0.6 9004109a 69.7 536 1.09004120a 71.2 506 0.9 9004142a 72.6 547 1.0 9004155a 75.4 435 0.99004193a 70.1 371 0.7 9004211a 72.6 557 1.1 9004226a 72.1 402 0.89004234a 72.8 548 1.0 9004239a 63.4 409 0.7 9004311a 68.1 506 0.99004319a 72.8 461 0.9 9004328a 68.8 478 0.9 9004346a 70.7 490 0.99004348a 71.5 508 0.9 9004354a 75.7 472 0.9 9004362a 56.6 388 0.69004367a 59.5 443 0.7 9004374a 50.9 523 0.7 9004376a 71.5 497 0.99004396a 72.8 516 1.0 9004411a 69.6 385 0.7 9004432a 57.3 490 0.79004443a 63.6 341 0.6

Example 12 Determining α-Terthiophene (α-Terthienyl) Levels in Extractsof Capsicum Varieties of the Instant Invention and Commercial Sources ofZeaxanthin and Lutein by GC-EI-MS and GC-PFPD

The analyses were performed on a Varian 3800 gas chromatograph in-linewith a Saturn 2000 ion trap mass spectrometer. The mass spectrometer wasoperated in the electron ionization mode with scanning from 40u to 650u.The NIST Standard Reference Database, version 1.6 was used for peakidentification. The GC-pulsed flame photometric detector was configuredfor sulfur-specific detection as per vendor specification. Dataacquisition utilized the Varian Saturn GC/MS data station (v5.51). Gaschromatography was performed on a Supelco MDN-5S fused silica capillarycolumn, 30 m×0.25 mm i.d., 0.25 um film (p/n 24384)). The column flowrate was 1.5 ml helium/minute; the injector temperature was 240° C.; thedetector temperature was 230° C.; the oven temperature program was 120°C. to 260° C. at 8° C./minute, hold at 260° C. for 4.5 minutes; theinjector split ratio was 1 for PFPD analysis and 20 for the GC-EI-MSruns. The injection volume was 0.5 μL.

A PFPD calibration curve for α-terthienyl (Aldrich,2,2′:5′,2″-Terthiophene, #311073, 99% purity) from 160 ng per ml to 5000ng per ml acetone was generated and used for subsequent quantitation.Oleoresin derived from the instant Capsicum plants, several commercialmarigold oleoresins and two nutritional supplement capsules were tested.The respective oleoresins were dissolved in acetone at 3300 microgram ofoleoresin per ml acetone prior to injection. The resulting sample arearesponse was converted to the corresponding α-terthienyl ppm value fromthe calibration curve by solving the second order polynomial equationthat was generated by a curve-fitting algorithm using Microsoft Excel2000. The results are listed below. FIG. 3 shows a chromatographiccomparison between the instant paprika oleoresin and a commercialmarigold oleoresin.

Sample α-terthienyl concentration Inventive Paprika Oleoresin Non-detectCommercial Zeaxanthin Capsule 1 1.4 microgram/capsule Commercial LuteinCapsule 2 2.2 microgram/capsule Commercial Marigold Extract 1 515 ppmCommercial Marigold Extract 2 1150 ppm Commercial Marigold Extract 3 760ppm Lower limit of detection (LOD) is 1.0 microgram/capsule for capsules

Example 13 Saponification of Oleoresin Derived from the Instant CapsicumVarieties and Preparation of Compositions with Higher Zeaxanthin Levels

Oleoresin from Example 4 (15.0 g), methanol (15 mL) and 45% aqueouspotassium hydroxide solution (6 mL) was combined in a 125 mL Erlenmeyerflask, equipped with Vigreux distilling column and magnetic stir bar andthe flask was wrapped in aluminum foil. The mixture was heated to refluxwith stirring for 1.5 hours. The dispersion was transferred to a 500 mLround bottom flask with soft water (30 mL). The methanol was removedfrom the round bottom flask with a rotary evaporator, and the solutionwas then transferred to a 600 mL beaker to which ethyl acetate was added(200 mL). The dispersion was stirred for 30 minutes and transferred toan aluminum-foil wrapped separatory funnel. The liquid phases were phaseseparated after a couple of hours. The water phase was washed with ethylacetate (200 mL) and the two decanted ethyl acetate fractions werecombined. Soft water (100 mL) was added to the combined ethyl acetatesolution and the liquid-liquid dispersion was stirred and neutralizedwith phosphoric acid. The dispersion was transferred to a separatoryfunnel and the water layer was decanted and removed. Heptane (100 mL)and soft water (25 mL) was added to the ethyl acetate layer thatremained in the separatory funnel and the dispersion was agitated. Thewater layer was removed and the organic phase was placed on a rotaryevaporator at 40-45° C. and 20 inches of pressure until enough ethylacetate was removed and solids began to form. The slurry was thenfiltered and this first crop of solids was rinsed with heptane. Theyield of the first crop of solids was 0.88 g with a purity ofapproximately 58% zeaxanthin. The filtrate solution was again placed onthe rotary evaporator and the rest of the solvent was removed, producingan oleoresin that contained additional solids. Heptane was added and thesolution was filtered to give a second crop of solids. The yield of thesecond crop of solids was 0.43 g with a purity of about 26% zeaxanthin.Evaporation of the heptane from the second filtrate produced anoleoresin with a color value (American Spice Trade Association Method20.1) of about 968.

Example 14 A Stabilized Oleoresin

The oleoresin from Example 4 is combined with natural tocopherols, andoptionally an edible oil, such that the final concentration ofzeaxanthin is 5% and the final concentration of added naturaltocopherols is 1%. The resulting fluid is encapsulated into gel capsulessuitable for human or animal consumption.

Example 15 A Stabilized Oleoresin

The oleoresin from Example 4 is combined with natural tocopherols,ascorbyl palmitate and optionally an edible oil, such that the finalconcentration of zeaxanthin is 5% and the final concentration of addednatural tocopherols and ascorbyl palmitate is 1%. The resulting fluid isencapsulated into gel capsules suitable for human or animal consumption.

Example 16 A Stabilized Formulation

The zeaxanthin solids from Example 13 are re-esterified with long chainfatty acids. The resulting zeaxanthin esters are formulated withascorbic acid, natural tocopherols, optionally a vegetable oil andoptionally rosemary extract to yield a finished product containing 5%zeaxanthin, 5% ascorbic acid, 5% added natural tocopherols and 0-5%rosemary extract. The resulting fluid is encapsulated into gel capsulessuitable for human or animal consumption.

Example 17 A Stabilized Formulation

The zeaxanthin solids from Example 13 are re-esterified with long chainfatty acids. The resulting zeaxanthin esters are formulated withascorbyl palmitate, natural tocopherols, optionally a vegetable oil andoptionally rosemary extract to yield a finished product containing 5%zeaxanthin, 1-5% ascorbyl palmitate, 5% added natural tocopherols and0-5% rosemary extract. The resulting fluid is encapsulated into gelcapsules suitable for human or animal consumption.

Example 18 A Stabilized Formulation

The zeaxanthin solids from Example 13 are dispersed into an edible oiland combined with ascorbic acid, natural tocopherols and optionallyrosemary extract to provide a product containing 20% zeaxanthin in free(non-esterified) form, 5% ascorbic acid, 5% tocopherol and 0-5% rosemaryextract.

Example 19 A Stabilized Formulation

The zeaxanthin solids from Example 13 are dispersed into an edible oiland combined with ascorbyl palmitate, natural tocopherols and optionallyrosemary extract to provide a product containing 20% zeaxanthin in free(non-esterified) form, 5% ascorbyl palmiate, 5% tocopherol and 0-5%rosemary extract.

Example 20 A Stabilized Formulation

The zeaxanthin solids from Example 13 or those solids that have beenfurther purified are dispersed into an edible oil and combined withlutein, ascorbic acid, natural tocopherols and optionally rosemaryextract to provide a product containing 0-20% lutein, 1-19% zeaxanthinin free (non-esterified) form, 1-5% ascorbic acid, 5% tocopherol and0-5% rosemary extract.

Example 21 A Stabilized Formulation

The zeaxanthin solids from Example 13 or those solids that have beenfurther purified are dispersed into an edible oil and combined withlutein, ascorbyl palmitate, natural tocopherols and optionally rosemaryextract to provide a product containing 0-20% lutein, 1-19% zeaxanthinin free (non-esterified) form, 1-5% ascorbyl palmitate, 5% tocopheroland 0-5% rosemary extract.

Example 22 A Stabilized Formulation

The oleoresin from Example 4 is combined with natural tocopherols, andolive extractives, such that the final concentration of zeaxanthin is 2%and the final concentration of added natural tocopherols is 1%. Theresulting fluid is encapsulated into gel capsules suitable for human oranimal consumption.

Example 23 ASTA Procedure (Adapted from ASTA method 20.1) for GroundPaprika for Samples with and without Seeds

Ground, ripe, dried fruit pod flesh with or without seeds of the instantCapsicum varieties (1.0 g) was weighed on a top loading balance and wasquantitatively transferred to a 125 ml Erlenmeyer flask. 50 ml ofacetone was added to the flask. The mixture was homogenized for 1minute. The solution was vacuum filtered through a Buchner funneldirectly into a 100 ml volumetric flask. All the color was rinsed out ofthe Erlenmeyer flask and the Buchner funnel with acetone, combined andbrought to a 100 ml total volume with acetone. A 1 ml sample waspipetted from the 100 ml volumetric into a 25 ml volumetric flask andbrought to a total of 25 ml with acetone. The spectrophotometer(Beckman, model: DU650) was set-up for a wavelength scan from 400 nm to550 nm and was zeroed using an acetone blank. A portion of the solutionin the 25 ml volumetric was transferred to the cell and a scan was runfrom 400 nm to 550 nm. The absorbance at 460 nm was determined. The ASTAwas calculated by the following equation:

ASTA=E1%1CM×16.4

The calculated percent zeaxanthin based on the ASTA value was calculatedusing the following formula:

% Calculated Zeaxanthin=(E1%1CM(Sample)/2340*)X% Zeaxanthin to totalCarotenoids.

*E1%1CM pure zeaxanthin=2340

Example 24 Concentration of Xanthophyll Pigments by Centrifugation

A quantity of oleoresin containing 6.25% total zeaxanthin, measured asthe free zeaxanthin, was processed through a high speed centrifuge.Operating conditions were varied to produce several concentratefractions, at least one of which contained 12.55% total zeaxanthin,measured as free zeaxanthin. The supernatant from the centrifugationcontained an enriched concentration of cryptoxanthin relative to thelevels present in the whole extract.

Example 25 Further Random Sampling of Production Fields

Samples of random individual Capsicum plants of the present inventionwere harvested. The pods were dehydrated in a laboratory dehydrator andwere subjected to analysis. The concentration of zeaxanthin in the driedfruit flesh was measured by the HPLC method described in Examples 26 and27. The percentage of zeaxanthin relative to total carotenoids, measuredin non-esterified forms, was also measured by the HPLC method describedin Example 28, and the analytical result for each sample is shown below.The ASTA value for each sample was also measured by the method describedin Example 23, and the corresponding mass percent zeaxanthin wascomputed from the measured ASTA value. The conversion of the ASTA valueinto the mass percent zeaxanthin was calculated by the proceduredescribed in Example 23.

Zeaxanthin Total in the dried Zeaxanthin pod with in the dried seeds podwith Zeaxanthin/ Wt % seeds Carotenoids Measured based on Sample (wt %)(area %) ASTA ASTA 9300014a 0.439 74 246 0.474 9300055a 0.549 66.7 3290.572 9300086a 0.464 64.9 299 0.506 9300100a 0.509 56.2 359 0.5269300124a 0.67 61.7 450 0.723 9300139a 0.64 61.7 408 0.656 9300140a 0.55465.4 351 0.598 9300247a 0.633 64.8 405 0.648 9300147a 0.584 79.5 2560.53 9300182a 0.497 65 340 0.576 9300254a 0.437 57.9 335 0.505 9300277a0.484 62.6 325 0.53 9300305a 0.401 62.2 354 0.574 9300326a 0.457 67.2272 0.476 9300345a 0.507 65.1 319 0.541 9300360a 0.462 66 318 0.5479300522a 0.494 57.6 374 0.561 9300369a 0.517 58.5 353 0.538 9300396a0.581 66.6 358 0.621 9300532a 0.606 66.5 365 0.632 9300571a 0.522 64.6356 0.599 9300575a 0.463 64.5 248 0.417 9300409a 0.472 62.7 327 0.5349300426a 0.574 57 366 0.544 9300434a 0.493 62.3 320 0.519 9300583a 0.74969.1 395 0.711 9300018a 0.417 60.1 304 0.476 9300047a 0.411 66.5 2440.423 9300222a 0.496 78.5 201 0.411 9300488a 0.506 63.9 255 0.4259300215a 0.471 63.8 295 0.490

Example 26 Saponification Procedure of Ground Capsicum with Seeds forHPLC Analysis

Ground, ripe, dried fruit pod flesh containing seeds of the instantCapsicum varieties (0.5 g) was weighed to the nearest tenth of amilligram on an analytical balance and was quantitatively transferred toa 125 ml Erlenmeyer flask. The flask was immediately covered withaluminum foil to reduce exposure to light. Butylated hydroxytoluene (0.2g, Sigma Chemical Company) and 1.5 g of sodium carbonate powder (AldrichChemical—A.C.S. reagent) were weighed and added to the Erlenmeyer flask.50.0 ml of methanol (Fisher Scientific—HPLC-grade) and 8 pellets (˜about0.8 g) of potassium hydroxide (VWR Intl.) were added to the Erlenmeyerflask. A stir bar was added to the solution and a Vigreux distillingcolumn was attached to the top of the Erlenmeyer flask. The solution wasplaced on a hot plate and refluxed on low heat (˜65° C.) with stirringfor 1 hour. Then the solution was taken off the hot plate and allowed tocool. A total of 1.2 ml of phosphoric acid (JT Baker—A.C.S. Reagent) wasadded to neutralize the solution. The solution was vacuum filteredthrough a Buchner funnel containing Celite® (Eagle Picher Filtration andMinerals, Reno, Nev.) directly into a 200 ml volumetric flask. All thecolor was rinsed out of the Erlenmeyer flask and the Buchner funnel withmethanol, combined and brought to a 200 ml total volume with methanol.After inverting the flask several times, the solution was poured into a3 cc syringe with a 0.45 micron PTFE Acrodisc® (Gelman) filter andinjected into an amber vial for HPLC analysis.

Example 27 Determination of Zeaxanthin Content (Wt %) by HPLC inCapsicum Pods with and without Seeds

The analyses were performed on a Waters 2695 (Milford, Mass. USA)separation system using Empower (Build 1154, Database version5.00.00.00) software installed on the data station. The chromatographicseparation was performed on a reverse-phase column (Waters Symmetry®C18, particle size 5 μm, 250 mm×4.6 mm). The eluent was a ternarygradient of methanol/water/acetone at 1.0 ml/min. The initialcomposition of the eluent was methanol-water-acetone (0:25:75, v/v/v).An initial linear gradient was applied for 15 minutes and yielded acomposition of methanol-water-acetone (20:5:75, v/v/v). This compositionwas held for 15 minutes, followed by another linear gradient for 5minutes to yield a composition of methanol-water-acetone (0:0:100,v/v/v) and held for 5 minutes. Another linear gradient was applied for 5minutes to initial conditions and held for 15 minutes before nextinjection. Compounds were detected photometrically (maxplot between 400nm-600 nm) on a Waters 2996 photodiode array detector using an injectionvolume of 20.0 μl. Zeaxanthin content was measured in reference to acalibration curve generated from a purchased authentic sample.Trans-zeaxanthin obtained from Indofine Chemical Company, Inc. and wasdissolved in 90% acetone/10% acetone containing 6% glacial acetic acid.This stock solution was diluted in acetone and run on a Beckman CoulterDU640 spectrophotometer at 452 nm. This absorbance was used with an E1%of 2340 to calculate the concentration of stock solution. The stocksolution was then diluted down with acetone to generate a 5-pointexternal calibration curve covering concentrations ranging from 4.0μg/ml-75.0 μg/ml with a linear fit. 9-cis-zeaxanthin was quantifiedusing the trans-zeaxanthin calibration curve, assuming a response factorof 1:1. Zeaxanthin contents are reported as a sum of all zeaxanthinisomers. A system check sample (DSM Zeaxanthin 20% FS; ProductCode:5002001; Lot: UE00303001) was run on the day of analysis at a levelbetween 25.0 μg/ml-45.0 μg/ml. Results were corrected only if checksample was not within 5% of the expected value.

Example 28 Determination of the Percentage of Zeaxanthin Relative toTotal Carotenoids (Area %) by HPLC for Capsicum Pods with and withoutSeeds

Analyses were performed on a Waters 2695 (Milford, Mass. USA) separationsystem using Empower (Build 1154, Database version 5.00.00.00) softwareinstalled on the data station. The chromatographic separation wasperformed on a reverse-phase column (Waters Symmetry® C18, particle size5 μm, 250 mm×4.6 mm). The eluent was a ternary gradient ofmethanol/water/acetone at 1.0 ml/min. The initial composition of theeluent was methanol-water-acetone (0:25:75, v/v/v). An initial lineargradient was applied for 15 minutes and yielded a composition ofmethanol-water-acetone (20:5:75, v/v/v). This composition was held for15 minutes, followed by another linear gradient for 5 minutes to yield acomposition of methanol-water-acetone (0:0:100, v/v/v) and held for 5minutes. Another linear gradient was applied for 5 minutes to initialconditions and held for 15 minutes before next injection. Compounds weredetected photometrically (maxplot between 400 nm-600 nm) on a Waters2996 photodiode array detector using an injection volume of 20.0 μl.Literature retention times and PDA spectra were used to identify some ofthe peaks (violoxanthin, antheraxanthin, 9-cis-zeaxanthin, cryptocapsin,α-cryptoxanthin, ζ-carotene). Other compounds were identified andcompared with standards from Carotenature (Lupsingen, Switzerland) andare listed as follows: capsorubin, capsanthin, trans-zeaxanthin, lutein,β-cryptoxanthin, α-carotene, trans-β-carotene and cis-β-carotene.

Example 29 Saponification Procedure of Ground Capsicum without Seeds forHPLC Analysis

Ripe, dried fruit pod flesh of the instant Capsicum varieties (0.5 g),without seeds, was ground and weighed to the nearest tenth of amilligram on an analytical balance and was quantitatively transferred toa 125 ml Erlenmeyer flask. The flask was immediately covered withaluminum foil to reduce exposure to light. Butylated hydroxytoluene (0.2g, Sigma Chemical Company) and 1.5 g of sodium carbonate powder (AldrichChemical—A.C.S. reagent) were weighed and added to the Erlenmeyer flask.50 milliliters of methanol (Fisher Scientific—HPLC-grade) and 8 pellets(˜about 0.8 g) of potassium hydroxide (VWR Intl.) were added to theErlenmeyer flask. A stir bar was added to the solution and a Vigreuxdistilling column was attached to the top of the Erlenmeyer flask. Thesolution was placed on a hot plate and refluxed on low heat (˜65° C.)with stirring for 1 hr. Then the solution was taken off the hot plateand allowed to cool. A total of 1.2 ml of phosphoric acid (JTBaker—A.C.S. Reagent) was added to neutralize the solution. The solutionwas vacuum filtered through a Buchner funnel containing Celite® (EaglePicher Filtration and Minerals, Reno, Nev.) directly into a 200 mlvolumetric flask. All the color was rinsed out of the Erlenmeyer flaskand the Buchner funnel with methanol, combined and brought to a 200 mltotal volume with methanol. After inverting the flask several times, thesolution was poured into a 3 cc syringe with a 0.45 micron PTFEAcrodisc® (Gelman) filter and injected into an amber vial for HPLCanalysis.

Example 30 Analysis of Capsicum without Seeds

Samples of random individual Capsicum plants of the instant inventionwere harvested. The pods were deseeded and dehydrated in a laboratorydehydrator and were treated along the lines of Example 29 for analysis.The concentration of zeaxanthin in the dried fruit flesh was measured bythe HPLC method described in Example 27. The percentage of zeaxanthinrelative to total carotenoids, measured in non-esterified forms, wasalso measured by the HPLC method described in Example 28, and theanalytical result for each sample is shown below.

Total Zeaxanthin in the dried pod Zeaxanthin/ without seeds CarotenoidsSample (wt %) (area %) 9300014b 0.55 65.9 9300055b 0.424 63.5 9300098b0.783 61.8 9300124b 0.849 59.6 9300043b 0.624 64.4 9300050b 0.783 59.29300080b 0.634 62.2 9300086b 0.517 57.5 9300289b 0.582 63.7 9300158b0.748 63.3 9300336b 0.844 64 9300358b 0.678 65.2 9300450b 0.742 64.89300473b 0.693 69.7 9300486b 0.711 64.8

Example 31 HPLC Separation of the Pigments

Both the red paprika and the instant orange paprika contain someunidentified pigments and their spectra indicate that there arealternate biosynthetic pathways operating in the red paprikas comparedto orange paprikas. Up to about 40% or more of the pigments in theyellows are currently not identified by HPLC. Because of thesesignificant differences, the yellow types are not further considered.

Many of the lesser pigments in the reds and oranges are intermediates inthe conversion of α- and β-carotene to their diol derivatives. Forexample, the cryptoxanthins are present in very differentconcentrations, as is violaxanthin, a precursor of capsorubin. Becausethese intermediates are present in relatively low amounts, they are notincluded in Table 3. below. Their relative amounts to total pigmentcontents may represent some variation in the actual maturity of thepods. The amounts are insufficient to affect the distinction between theclasses, and may portray biochemical differences between the red-fruitedand orange-fruited strains.

TABLE 3 Key pigment ratios distinguishing orange-colored fromred-colored paprikas. Zeaxanthin Capsanthin + α-Carotene/β- (Trans +Cis) Lutein Capsorubin Lutein/Zeaxanthin Carotene Capsanthin/ZeaxanthinASTA % % % Ratio Ratio Ratio Reds Mean = 464.64 13.04 5.83 33.17 0.500.02 2.52 STD = 4.69 0.71 3.13 0.17 0.01 0.79 Oranges Mean = 360.7068.48 0.92 4.36 0.02 0.05 0.06 STD = 5.12 0.65 2.04 0.01 0.10 0.03Table 3. shows the key differences between the level of pigments inrepresentative types of red and orange paprikas. It will be noted thatcapsorubin and capsanthin predominate in the red-fruited strains,whereas zeaxanthin predominates in the orange-fruited strains and thelevel of the red pigments is about half or less of that in the redstrains. The zeaxanthin level observed in the orange-fruited strains is˜4-5 times higher than that observed in the red-fruited strains on arelative basis. The lutein level observed in the orange-fruited strainsis ˜5-6 times less than in the red strains on a relative basis. The sumof capsanthin plus capsorubin observed in the orange-fruited strains is˜7-8 times less than in reds on a relative basis. The ratio of lutein tozeaxanthin observed in the orange-fruited strains is ˜25 times less thanin reds on a relative basis. The ratio of α-carotene to β-caroteneobserved in the orange-fruited strains is ˜2 times more than inred-fruited strains on a relative basis. The ratio of capsanthin tozeaxanthin in the orange-fruited strains is ˜42 times less than in thered strains on a relative basis. The analysis affirms that there aredifferences in the carotenoid biosynthetic pathways between the instantorange paprika and red paprika strains described herein.

It should be noted that ASTA is used as a proxy for molar or weightratios of pigments. ASTA utilizes the absorbance of a solution of theextract at a wavelength of 460 nm. The orange paprikas have a lambdamaximum at 454-455 nm, whereas the red paprikas have a lambda max closeto 460 nm. Therefore, the ASTA of equal pigment content is somewhatlower for an orange paprika than a red paprika. From the standpoint ofseparating the genotypes, this does not make a difference. It shouldalso be noted that in field sampling, there is always a variation inASTA observed on a single plant and between different plants of the samestrain. This is due to variations in maturity, disease and otherstresses, as well as field soil differences.

Table 3. presents reasonable average values for ASTAs and HPLC ratiosfor the purpose of demonstrating the distinct biochemical differencesbetween the red-fruited strains and the instant orange-fruited strains.Individual pods from the same or different plants may have differentpigment ratios and ASTAs. The averages show what may be reasonablyexpected from a normal crop derived from these paprikas.

The instant orange paprikas were developed by careful hybridization,selection of superior plants which, through recombination of genes orpromoters, modified the carotenoid pathway without reducing the pigmentcontent. The orange strains are comparable to commercial red paprikas inASTA, but with a novel pigment profile, high in zeaxanthin, which has adistinctly different absorption spectrum than the reds. Therefore astrain or strains of a Capsicum annuum paprika type cultivar is thepreferred type of Capsicum as the source of zeaxanthin. It iscommercially attractive if zeaxanthin is present at more than about 50%of the area count of the total pigments. As the area count % increasesto 65%, further to 75%, and even further to 80%, the cost of thezeaxanthin is reduced. It is also the preferred source of an oleoresinrich in zeaxanthin.

DEPOSIT INFORMATION

Applicant has deposited at least 2500 seeds of Capsicum annuum NM 1441disclosed herein with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209 USA under the terms ofthe Budapest Treaty. The accession number for the deposit is ATCCAccession No. PTA-10729 and the date of deposit is Mar. 25, 2010. Accessto this deposit will be available during the pendency of the applicationto the Commissioner of Patents and Trademarks and persons determined bythe Commissioner to be entitled thereto upon request.

In accord with 37 CFR §1.808, the Applicant declares that allrestrictions imposed by the depositor on the availability to the publicof the deposited material will be irrevocably removed upon the grantingof the patent. Moreover, Applicant reserves the right to contract withthe depository to require that samples of a deposited biologicalmaterial shall be furnished only if a request for a sample, made duringthe term of the patent:

(1) Is in writing or other tangible form and dated;(2) Contains the name and address of the requesting party and theaccession number of the deposit; and(3) Is communicated in writing by the depository to the depositor alongwith the date on which the sample was furnished and the name and addressof the party to whom the sample was furnished.

REFERENCES

-   Abel, R. Jr., 2004, The Eye Care Revolution: Prevent and Reverse    Common Vision Problems, Kensington Publishing Corp., NY, N.Y. ISBN    0-7582-0622-4, p. 159.-   Abellan-Palazon, M., 2001, Effect of Titanium Ascorbate Treatment on    Red and Yellow Pigment Composition of Paprika Cultivars, Acta    Alimentaria, Vol. 30 (2), pp. 159-171.-   Ahmed S. et al., 2005, The Macular Xanthophylls. Sury Ophthalmol.,    50(2), pp 183-93.-   Almela, L. et al., 1991, Carotenoid Composition of New Cultivar of    Red Pepper for Paprika, J. Agric. Food Chem., 39, pp. 1606-1609.-   Almela, L. et al., 1996, Changes in Pigments, Chlorophyllase    Activity and Chloroplast Ultrastructure in Ripening Pepper for    Paprika, J. Agric. Food Chem. 44, pp. 1704-1711.-   Alves-Rodrigues A, and Shao A., 2004, The Science Behind Lutein.    Toxicol Lett. April 15, 150(1), pp. 57-83.-   Antony, J. I. X and Shankaranarayana, M. L., 2001, Lutein—A Natural    Colorant and a Phytonutrient For Eye Health Protection, The World of    Food Ingredients, April/May, pp. 64-67.-   Aranson, J. T. et al., 1995, Fate of Phototoxic Terthiophene    Insecticides in Organisms and the Environment in Light-Activated    Pest Control, ACS Symposium Series 616, J. R. Heitz and K. R.    Downum, eds., American Chemical Society, Washington D.C., pp.    143-151.-   Banaras, M. et al., 1994, Relationship of Physical Properties to    Postharvest Water Loss on Pepper Fruits (Capsicum annuum L.),    Pak. J. Bot., 26(2), pp. 321-326.-   Beatty S. et al., 2004, Macular Pigment Optical Density and Its    Relationship With Serum and Dietary Levels of Lutein and Zeaxanthin.    Arch Biochem Biophys., 430(1), pp. 70-6.-   Biacs, P. et al., 1993, Carotenoids and Carotenoid Esters from New    Cross-Cultivars of Paprika, J. Agric. Food Chem., 41, pp. 1864-1867.-   Biacs, P. and Daood, H., 1994, High Performance Liquid    Chromatography with Photodiode-array Detection of Carotenoids and    Carotenoid Esters in Fruits and Vegetables, J. Plant Physiol. Vol.    143, pp. 520-525-   Bouvier F, et al., 1994, “Xanthophyll Biosynthesis in Chromoplasts:    Isolation and Molecular Cloning of An Enzyme Catalyzing The    Conversion of 5,6-Epoxycarotenoid Into Ketocarotenoid,” Plant    Journal 6, pp. 45-54.-   Bowen, P. E., et al., 2002, Esterification Does Not Impair Lutein    Bioavailability in Humans, J. Nutr. 132, pp. 3668-3673.-   Breithaupt, Dietmar E., et al.; British Journal of Nutrition,    (2004), 91, pp. 707-713.-   Breithaupt, Dietmar E. & Schlatterer, Jorg; Eur Food Res    Technol, (2005) 220, pp. 648-652.-   Breithaupt, D. E., and Bamedi, A., 2001, Carotenoid Esters in    Vegetables and Fruits: A Screening with Emphasis on β-Cryptoxanthin    Esters, J. Agric. Food Chem., 49, pp. 2064-2070.-   Brown L. et al., 1999, A Prospective Study of Carotenoid Intake and    Risk of Cataract Extraction in US Men. Am J Clin Nutr., 70(4), pp.    517-24.-   Camara, Bilal & Moneger, Rene; Phytochemistry, 1978, Vol. 17, pp.    91-93-   Connor S. et al., 2004, Diets Lower in Folic Acid and Carotenoids    Are Associated with The Coronary Disease Epidemic in Central and    Eastern Europe. J Am Diet Assoc. 104(12): pp. 1793-9.-   Davies, N. P. et al., 2004, Macular Pigments: Their Characterization    and Putative Role, Prog. Ret. Eye Res. 23, pp. 533-559.-   Deli, J. et al., 1992, Carotenoid Composition in the Fruits of Black    Paprika (Capsicum annuum Variety longum nigrum) during Ripening, J.    Agric. Food Chem., 40, pp. 2076.-   Deli, J. et al., 1996, Carotenoid Composition in the Fruits of    Capsicum annuum Cv. Szentesi Kosszarvu during Ripening, J. Agric.    Food Chem., 44, pp. 711-716.-   Deli, J., and G. Toth, 1997, Carotenoid Composition of The Fruits of    Capsicum annuum Cv. Bovet 4 During Ripening,” Z Lebensm Unters    Forsch A, 205, pp. 388-391.-   Deli, J. et al., 2001, Carotenoid Composition in the Fruits of Red    Paprika (Capsicum annuum var. lycospersiciforme rubrum) During    Ripening: Biosynthesis of Carotenoids in Red Paprika,” J. Agric.    Food Chem., 49, pp. 1517-1523.-   Deruere, J., et al., 1994, The Plant Cell, Vol. 6, pp. 119-133-   Downum, K. R. and J. Wen, 1995, “The Occurrence of Photosensitizers    Among Higher Plants”, in Light-Activated Pest Control, ACS Symposium    Series 616, J. R. Heitz and K. R. Downum, eds., American Chemical    Society, Washington D.C., pp. 135-143.-   Englert, G. et al, 1991, HeIv. Chim. Acta., 74, pp. 969-982.-   Evans et al., 1993, Handbook of Plant Cell Culture, MacMillian    Publishing Co., NY-   Fisher, C. and Kocis, J. A., 1987, J. Agric. Food Chem. 35, pp.    55-57.-   Gelvin et al., 1990, Plant Molecular Biology Manual, Kluwer Academic    Publishers-   Green et al., 1987. Plant Tissue & Cell Culture, Academic Press, NY-   Hausen, B. M., and B. Helmke, 1995, “Butenylbithiophene,    α-terthienyl and Hydroxytremetone As Contact Allergens in Cultivars    of Marigold (Tagetes sp.),” Cont. Derm. 33, pp. 33-37.-   Hirschberg, J., 2001, “Carotenoid Biosynthesis in Flowering Plants,”    Current Opinions in Plant Biology 4(3), pp. 210-218.-   Hornero-Mendez, D. et al., 2002, “Characterization of Carotenoid    High-Producing Capsicum annuum Cultivars Selected for Paprika    Production,” J. Agric. Food Chem., 50, pp. 5711-5716.-   Ishida, B. K., et al, 2005, Assessing Bioavailability of cis-vs    trans-lycopene isomers in Tangerine and Red Tomatoes, Carotenoid    Science, Vol. 9, pp. 92.-   Isler, 0. et al, 1956, HeIv. Cim. Acta., 39, p. 249-   Ito Y, et al, 2005, JACC Study Group. Lung Cancer Mortality and    Serum Levels of Carotenoids, Retinol, Tocopherols, and Folic Acid in    Men and Women: A Case-Control Study Nested in The JACC Study. J.    Epidemiol. Suppl 2:S140-9. PMID: 16127226-   Kahl et al., (1995) World Journal of Microbiology and Biotechnology    11, pp. 449-460-   Karrer, P. and Jucker, E., 1950, Carotenoids, Elsevier Publ. Co.,    Inc., Amsterdam, pp. 38-42, 180 ff-   Khachik, F. et al., 1992, Isolation and Structural Elucidation of    the Geometrical Isomers of Lutein and Zeaxanthin in Extract from    Human Plasma, J. Chromatrogr. Biomed. Appl. 582, pp. 153-166.-   Khachik, F., et al., 1995, Lutein, Lycopene, and Their Oxidative    Metabolites in Chemo-prevention of Cancer, J. Cell. Biochem., Suppl.    22, pp. 236-246.-   Khachik F, et al., 1997, Identification of Lutein and Zeaxanthin    Oxidation Products in Human and Monkey Retinas. Invest Ophthalmol    Vis Sci., pp. 1802-11.-   Khachik, F. et al., 1999, Dietary Carotenoids and their Metabolites    as Potentially Useful Chemoprotective Agents against Cancer, in    Antioxidant Food Supplements in Human Health, L. Packer, M.    Hiramatsu and T. Yoshikawa, eds., Academic Press, NY, pp. 203-229.-   Klee et al., 1987, Ann. Rev. of Plant Phys. 38, pp. 467-486-   Kohlmeier, L. et al., 1995, Am. J. Clin. Nutr. 62, pp. 137-146-   Levy, Joseph, et al., 1995, Lycopene Is a More Potent Inhibitor Of    Human Cancer Cell Proliferation Than Either α-Carotene or    β-Carotene,” Nutrition & Cancer, pp 257-266.-   Lutein and Zeaxanthin Scientific Review, Roche Vitamins Technical    Publication HHN-1382/0800-   Lyle B J, et al., 1999, Antioxidant Intake and Risk of Incident    Age-Related Nuclear Cataracts in The Beaver Dam Eye Study., Am J.    Epidemiol., pp. 801-9.-   Matus et al., 1991, Carotenoid Composition of Yellow Pepper during    Ripening: Isolation of β-Cryptoxanthin 5,6-Epoxide, J. Agric. Food    Chem., 39, pp. 1907-1914.-   Minguez-Mosquera, M. I. et al., 1993, Effect of Processing of    Paprika on the Main Carotenes and Esterified Xanthophylls Present in    the Fresh Fruit, J. Agric. Food Chem., 41, pp. 2120-2124.-   Minguez-Mosquera, M. I. et al., 1994, Comparative Study of the    Effect of Paprika Processing on the Carotenoids in Peppers (Capsicum    annuum) of the Bola and Agridulce Varieties, J. Agric. Food Chem.,    42, 1555-1560.-   Minguez-Mosquera, M. and Hornero-Mendez, D., 1994, Changes in    Carotenoid Esterification during the Fruit Ripening of Capsicum    annuum Cv. Bola, J. Agric. Food Chem., 42, pp. 640-644.-   Minguez-Mosquera, M. and Hornero-Mendez, D., 1993 Separation and    Quantification of the Carotenoid Pigments in Red Peppers (Capsicum    annuum L.), Paprika, and Oleoresin by Reversed-Phase HPLC, J. Agric.    Food Chem., 41, pp. 1616-1620.-   Muller, H., 1997, Determination of the carotenoid content in    selected vegetables and fruit by HPLC and Photodiode array    detection, Z. Lebensm Unters Forsch A., 204, pp. 88-94-   Murakoshi et al., 1992, Cancer Res., 52, pp. 6583-6587-   Nys, Y. Arch. Geflugelk, 2000, 64 (2), pp. 45-54-   Osganina S., et al., 2003, Dietary Carotenoids and Risk of Coronary    Artery Disease in Women, Am. J. Clin. Nutr. 2003 June; 77(6), pp.    1390-1393.-   Packer. L. et al., (Editors), 1999, Antioxident Food Supplements in    Human Health, Academic Press, NY, pp 223-226.-   Pattison, D., 2005, American Journal of Clinical Nutrition, Pub. by    American Society for Clinical Nutrition, Vol. 82, No. 2, pp. 451-455-   Rahman, R. M. M., and K. A. Buckle, 1980, “Pigment Changes in    Capsicum Cultivars During Maturation and Ripening”, J. Fd. Technol.,    15, pp. 241-249.-   Ribaya-Mercado J. et al., 2004, Lutein and Zeaxanthin and Their    Potential Roles in Disease Prevention. J. Am. Coll. Nutr., 23(6    Suppl), pp. 567S-587S.-   Rock C. et al., 2005, Plasma Carotenoids and Recurrence-free    Survival in Women with a History of Breast Cancer. J. Clin. Oncol.,    pp. 6631-8.-   Russo, V. M. and Howard, L. R. J. Sci. Food Agric. 82: 615-624.-   Seddon, J. M. et al., 1994, Dietary Carotenoids, Vitamins A, C, and    E, and Advanced Age-Related Macular Degeneration,” J. Am. Med.    Assoc. 272(9), pp. 1413-1420.-   Sommerburg, Olaf, et al.; Br F Ophthamol, 1999, 82, pp. 907-910.-   Stahl W., 2005, Macular Carotenoids: Lutein and Zeaxanthin. Dev    Ophthalmol. 38, pp. 70-88.-   Stahl W. and Sies H., 2005, Bioactivity and Protective effects of    Natural Carotenoids. Biochim Biophys Acta., 1740(2), pp. 101-7. Epub    2004 Dec. 28.-   Stringham J. and Hammond B. Jr., 2005, Dietary Lutein and    Zeaxanthin: Possible Effects on Visual Function, Nutr. Rev. 63(2),    pp. 59-64.-   Tanaka et al., 1994, Carcinogenesis 15, pp. 15-19-   Topuz, A. and Ozdemir, F., 2003, Influences of γ-Irradiation and    Storage on the Carotenoids of Sun-Dried and Dehydrated Paprika, J.    Agric. Food Chem., 51, pp. 4972-4977.-   Updike, A. A., and Schwartz, S. J., 2003, Thermal Processing of    Vegetables Increases the Cis Isomers of Lutein and Zeaxanthin, J.    Agric. Food Chem. 51, pp. 6184-6190.-   Vasil., 1984, Cell Culture and Somatic Cell Genetics of Plants, Vol    I, II, Ill, Laboratory Procedures and Their Applications, Academic    Press, NY-   Weissbach and Weissbach, 1989, Methods for Plant Molecular Biology,    Academic Press-   Weller, P. and Breithaupt, D., 2003, Identification and    Quantification of Zeaxanthin Esters in Plants Using Liquid    Chromatography—Mass Spectrometry, J. Agric. Food Chem., 51, pp.    7044-7049.-   Yin et al., 2004, Transgene Inheritance In Plants. J. Appl. Genet.    45(2), pp. 127-144 and references therein.-   Zechmeister, L., 1962, Cis-Trans Isomeric Carotenoids Vitamins A and    Arylpolyenes, Academic Press, pp. 46-57.-   Zhou, L. et al., 1999, The Identification of Dipalmityl Zeaxanthin    as the Major Carotenoid in Gou Qi Zi by High Pressure Liquid    Chromatography and Mass Spectrometry, J. Ocular Pharm. and Therap.,    15(6), pp. 557-565.

1. A Capsicum annuum paprika plant crude oleoresin obtained from anisolated Capsicum annuum paprika plant obtained by inbreeding Capsicumannuum NM 1441, deposited at the American Type Culture Collection (ATCC)under accession number PTA-10729, and selecting for a plant whichproduces orange ripe fruit pods, and which plant exhibits zeaxanthin inthe dried ripe fruit pod flesh, wherein the mass of zeaxanthin, whenmeasured in non-esterified form, is greater than 0.4% of the mass oftotal dried ripe fruit pod flesh and wherein zeaxanthin is the dominantcarotenoid, when measured in non-esterified forms, wherein thepercentage of zeaxanthin relative to total carotenoids [masszeaxanthin/(mass zeaxanthin plus mass of other carotenoids)×100] in thecrude oleoresin is greater than 50% when measured in non-esterifiedforms by High Performance Liquid Chromatography (HPLC) area count, andwherein the zeaxanthin is present in the crude oleoresin in an amount of6.7% when measured in non-esterified form by HPLC.
 2. The Capsicumannuum paprika plant crude oleoresin of claim 1, which exhibits an ASTAvalue in a range from 341 to
 584. 3. The Capsicum annuum paprika plantcrude oleoresin of claim 1, further comprising cryptoxanthin, lutein andother carotenoids.
 4. The Capsicum annuum paprika plant crude oleoresinof claim 1, wherein the oleoresin is substantially free fromterthiophenes.
 5. The Capsicum annuum paprika plant crude oleoresin ofclaim 1, which is combined with natural and synthetic antioxidants. 6.The Capsicum annuum paprika plant crude oleoresin of claim 1, which isin the form of an emulsion.
 7. The Capsicum annuum paprika plant crudeoleoresin of claim 1, which is dispersed on solids suitable fornutritional supplement, food, beverage, cosmetic or pharmaceuticalapplications.
 8. The Capsicum annuum paprika plant crude oleoresin ofclaim 1 in the form of beadlets suitable for nutritional supplement,food, beverage, sometic or pharmaceutical applications.
 9. The Capsicumannuum paprika plant crude oleoresin of claim 1, which is combined withone or more other natural and/or synthetic carotenoids to provide mixedcarotenoid compositions.
 10. The Capsicum annuum paprika plant crudeoleoresin of claim 1, which is combined with natural tocopherols, andoptionally an edible oil, wherein the final concentration of zeaxanthinis 5% and the final concentration of added natural tocopherols is 1%,and which is encapsulated into gel capsules suitable for human or animalconsumption.
 11. The Capsicum annuum paprika plant crude oleoresin ofclaim 1, which is combined with natural tocopherols, and optionally anedible oil, natural tocopherols, ascorbyl palmitate and optionally anedible oil, wherein the final concentration of zeaxanthin is 5% and thefinal concentration of added natural tocopherols and ascorbyl palmitateis 1%, and which is encapsulated into gel capsules suitable for human oranimal consumption.
 12. The Capsicum annuum paprika plant crudeoleoresin of claim 1, which is combined with food grade emulsifiers. 13.The Capsicum annuum paprika plant crude oleoresin of claim 12 which iscomprised in beverages and emulsion-based foods.
 14. The Capsicum annuumpaprika plant crude oleoresin of claim 9 in the form of a nutritional orfeed supplement, food or feed colorant, or food or feed additive. 15.The Capsicum annuum paprika plant crude oleoresin of claim 1 in the formof a food colorant.